CA2139105A1 - Angiotensin iv peptides and receptor - Google Patents

Abstract

A unique and novel angioten-sin AT4 receptor and AIV ligand sys-tem for binding a small N-terminal hexapeptide fragment of Angiotensin II (referred to as AIV, with amino ac-id sequence Val1-Tyr2-Ile3-His4-Pro5-Phe6) is disclosed. AIV ligand binds satu-rably, reversibly, specifically, and with high affinity to membrane AT4 receptors in a variety of tissues, in-cluding heart, lung, kidney, aorta, brain, liver, and uterus, from many animal species. The AT4 receptor is pharmacologically distinct from classic angiotensin receptors (AT1 or AT2). The system employs AIV
or C-terminally truncated or ex-tended AIV-like peptides (e.g. VY-IHPFX) as the signaling agent, and the AT4 plasma membrane receptor as the detection mechanism. The an-giotensin AT4 receptor and receptor fragments (including the receptor binding site domain) are capable of binding a VYIHPF angiotensin AIV N-terminal peptide but not an angiotensin AII or AIII N-terminal peptide, i.e., DRVYIHPF or RVYIHPF, respectively. Also disclosed are processes for isolating angiotensin AT4 receptor and AIV angiotensinase, identifying angiotensin AIV agonists and antagonists, and constructing diagnostic assays to specifically measure AIV and AI-specific angiotensinase in biological fluids.

Description

' 'O 94/00492 2 1 3 9 1 0 S PCr/US93/06038 ANGIOTENSIN IV ~ )ES AND RECEPTOR
Field of the Invention This invention relates to the polypeptide ligand VYIHPF (angiotensin IV or AIV) and to related peptide ligands and poly~minoa~.id ligands that bind to, activate S and/or antagonize a novel angiotensin AT4 receptor. The ligands comprise at least three of the N-terminal arnino acids of AIV, or AT4 receptor binding equivalents or analogs thereof. Engagement of the receptor by its ligand triggers acute physiological effects (e.g., vasodilation) and long-term effects in cells (e.g., hypertrophic growth).
Background of the Invention The renin-angiotensin system has wide-ranging actions on numerous tissues in the body affecting blood pressure (pressor activity) and cardiovascular and electrolyte homeostasis. It is ~;u~ ly believed that angiotensins AII and AIII are derived via enzymatic cleavage in the cascade depicted in Figure 1, steps 1, 2, and 3 (l).
(Numbering herein of the amino acid residues in AI, AII, Am, and AIV is according 15 to that appearing in Figure 1.) The renin-angiotensin cascade is thought to begin with the action of renin on angiotensinogen to release angiotensin I (AI), a biologically inactive decapeptide. Angiotensin II (AII), the bioactive Oclai)eplide~ is thought to be formed by the action of angiotensin converting enzyme (ACE) on circ ll~ting AI (2).
Des-AspAII (Angiotensin III; Am) is derived from AII, and certain reports have 20 suggested possible activities for Am in the adrenal gland (3) and brain (4). It has been reported that AII and AIII are inactivated by el~ylllalic degradation through a series of smaller inactive fr~gm~nts (5). F~ smaller than Am have been thought, for the most part, to be biologically inactive and of little physiological ~ignifis~nce (6). This assumption has been based on the lack of pressor and certain 25 endocrine activities (i.e., aldosterone release) of small angiotensin fragrnlont~ (7) and the finding that N-terminal deleted fraem~nt~ i.e., smaller than AIII, reportedly

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exhibit low binding affinity for angiotensin AI or AII receptors (known as AT1 and AT2, respectively) as determined in radiolabeled ligand studies (8).
Certain studies have used AII(3 8) as one of several controls in structure-activity studies of AT1 and AT2 receptors (9,10). An AII receptor having components with molecular weights of 60-64kDa and 112-115kDa has reportedly been cloned from adrenal cortical cells as well as rat smooth muscle (11).
In general, AII(3 8~ has been found to be much less active than AII or Am with regard to typical angiotensin-dependent pressor activity or stim~ fing water intake (9,10,12). However, certain reports have suggested that AII(3 8), while having little pressor activity or ability to stim~ te aldosterone release, may under certain circ~ ces inhibit renin release from kidney (12,13). Haberl et al. (14) reported a possible effect of AII(3 8~ on endothelium-dep.onrlçnt dilation in rabbit brain.Braszko etal. (15,16) reported possible effects of AII(3 8~ or AII(3 7~ on motoractivity, memory, and learning when ~minict~red intracerebru~entricularly (icv) into rat brain and s~lEEested that these effects should be considered "unspecific," i.e., not metli~ted by receptors (Braszko et al. (17), p. 195).
The angiotensin field has often been fraught with complexity and conflicting i,~"l,aLion, particularly with regard to the levels of di~l~"l AII and AIII peptides required to elicit certain cellular responses, the concentrations predicted from receptor binding studies to be biologically active, and the levels of angiotensin peptides that may be measured in biological fluids. It has been reported that AII and AIII areremoved from, or destroyed in, circulation by enzymatic hydrolysis. Biological half-lives of the di~ "l metabolic fr~Em~ntc are reportedly quite short. Semple and co-workers (18) reportedly detected AIII, AII(3 8~, and AII(4 8~ in arterial and venous blood in man with half-lives for AII, Am, AII(3 8~, and AII(4 8~ of 4.4, 2.0, 1.9, and 2.4 minlltes~ res~e~ ely. Blumberg et al. (19) reported that during transit through the kidney 72-76% of AI and AII and 89% of Am was metabolized.
Confusion has existed in the art as to how metabolic products of AII and AIII
can exhibit certain biological activities (e.g., inhibition of renin release andenhAnce~ of cognitive function), while failing to bind to AI or AII recep~o~.
Fr~Em~.ntc of AII smaller than Am, e.g., AII(3-8) and other smaller fr~m~ntc have not been reported to have specific saturable binding sites in tissues, and ,~ce~,lo,~ for these fraEm~ntc have not been identified previously. The present invention provides partial explanation for certain previous confusing and contradictory fintlingc, and provides novel AIV receptors (AT4), AIV ligands, peptides, analogs, agonists andantagonists that bind specifically to the AT4 receptor and not to AI (AT1) or AII

~vo 94/00492 2 ~ ~ PCr/US93/06038 10 ~

(AT2) receptors. The AIV peptides and the AT4 receptor are labile and subject toproteolytic degradation. In other aspects, the invention provides a specific angiotçnin~e enzyme that converts AII or AIII peptides to AIV peptides in a novel pathway.
Summary of the Invention The discovery, herein, of a unique and novel angiotensin AIV receptor (AT4) and AIV ligand system for binding a small N-terminal h~;Aapeplide fragment of Angiotensin II (referred to herein as AIV, with amino acid sequence Vall-Tyr2-Ile3-His4-Pro5-Phe6) provides partial explanation for confusion in the prior art. AIV binds saturably, reversibly, spe~ifis~lly, and with high affinity to me~ e AT4 receptors in a variety of tissues and from many animal species. The AT4 receptor is pharmacologically distinct from classic angiotensin leceplo~ (AT1 or AT2) in that the AT4 leceptor displays no specificity for classic agonists (AII and AIII) and antagonists (Sarl,Ile8-AlI). Thus, the disclosure details the pharmacological and bioch~mic~l characterization of a newly discovered branch of the renin-angiotensin system that employs an AIV ligand as the ~ ling agent, and theAT4 plasma membrane receptor as the detection mech~ni~m Angiotensin AIV appears to specifically mobilize calcium in vascular endothelial cells where AIV binding is evident. Binding to the endothelial AT4 receptor appears to trigger cellular proliferation. Binding of AIV to AT4 receptors in kidney and brain increases blood flow. In addition, binding of AIV to AT4 receptors in the brain f~.ilit~tes learning and memory retention. AIV has also been shown to block the hypertrophic action of AII on cardiocytes despite its inability to bind AT2 receptors. Since cardiocytes possess large numbers of AT4 receptors this action of AIV is most likely direct. Thus, in certain ~ pecl~ the action of AIV appears toneutralize, or act in apposition to the actions of AII and Am.
The invention provides an angiotensin AT4 receptor and receptor fragments (inclllding the receptor binding site domain) that are capable of binding a VYIHPF
angiotensin AIV N-terminal peptide, and related AIV ligands, but do not bind an angiotensin AII or AIII N-terminal peptide, i.e., DRVYIHPF or RVYIHPF, respectively. The AT4 receptor from adrenal cortical cells has a molecular size of about 140kD to about 150kD on SDS-PAGE following crosslinking a Kd Of about 0.5nM for AIV peptides, and is widely cAplessed on the surface of adrenal cortical and me~ ry tissues in many ,.. ~.. ~li~n species. The receptor is t;A~Iessed in all 35 important organs and tissues in~ ling heart, lung, kidney, aorta, brain, liver, and uterus.

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The invention further provides processes for identifying angiotensin AIV
agonists and antagonists, and constructing diagnostic assays to specifically measure AIV and AT4 receptors.
Brief Description of the Drawin~s FIGURE 1 is a sçhPm~tic diagrarn depicting the amino acid sequence of angiotensinogen and its conversion by renin to AI, by angiotensin converting enzyme (ACE) to AII, by angiopeptidase to AIII, and by a novel AIV angiotçncin~ce, herein disclosed, to angiotensin AIV (AIV).
FIG~RE 2A is a graphical leyresenlalion of the results of equilibrium binding studies of l25I-radiolabeled AIV to AT4 lCCcylol~, isolated from bovine adrenal cortical llltlll~l ~nes; as described in F.Y ~ ~ .ple 1.
FIGURE 2B depicts graphically the structural requilclllcllls and specificity forbinding of AIV ligand to the AT4 receptor from rabbit cardiac myocyte lllclll~l~nes;
as described in F.Y~mple 1.
FIGURE3 COllly~cS AT2 and AT4 receptor loc~li7~fion in the Habenula region of the brain using receptor autoradiography with 125I-Sarl,Ile8-AII to localize AT2 receptors, and l25I-AIV to localize AT4 leceplol~, as described in Example 2.
Panel A shows binding of l25I-AIV to cells in the habenula, th~l~mlls~ cerebral cortex and hippocampus of guinea pig brain. Panel B shows that the binding of 125I-AIV is spe~ ific~lly col.,yc~ ely inhibited by lOOnM non-labeled AIV co---yc~i~or. Panel C
shows that binding of 125I-AIV is not co...pc~ ely inhibited by l OOnM Sarl,Ile8-AII.
Panel D shows a pattern of binding of 125I-Sarl,Ile8-AlI to AT2 receptors that is di~clcnl from the pattern observed with l25I-AIV in Panel A. Panel E shows that binding of 125I-Sarl,Ile8-AII is specifically inhibited by lOOnM of non-labeled AII
25 coyc~ilor Panel F shows that binding of 125I-Sarl,Ile8-AII is not inhibited by lOOnM non-labeled AIV colllyctiLor. Panel G shows a "pseudo-color" photograph of12~I-AIV binding. Panel H shows a "pseudo-color photograph of 125I-Sarl,Ile8-AIIbinding. Panel I shows a photomicrograph of a histology slide of a serial section of the same tissue as in Panels A-I.
FIGURE 4 graphically depicts the pe.ccll~age change in renal blood flow after infusion of lOOpmol of AIV (n=13 ~"~yclinlcllls); 0.15M saline (n=9); lOOpmol ofD-Vall-AIV (i.e., AIV with a D-valine residue in the 1 position); or lOOpmol of AII
(n=8) into the renal artery at a rate of 25ml/min, as described in Example 6.
FIGURES 5A and 5B are graphical epresclllalions of changes in blood flow that result from binding of agonist, LyslAIV, to AT4 receptors in kidney, without changes in systemic blood ples~-lre, as described in Example 4. Figure 5A shows vo 94/00492 PCr/US93/06038 changes in arterial blood pressure following ~minictration of LyslAIV at 100pmole/25ml/min (open circles) or saline control (closed circles). Figure 5B shows changes in renal blood flow following ~mini~tration of LyslAIV at 100pmole/25 I/min (open circles) or saline control (closed circles).
FIGURES 6A and 6B are graphical leplese,llaLions showing r.h~nges in blood flow that result from ~tlmini~tering di~rellL doses of an agonist NorLeulAIV (i.e., NorLeuYIHPF) that binds to AT4 lecel,tol~ in kidney, without changes in systemicblood pressule~ as described in Example4. A Lllt;l~t;LItically effective dose for increasing renal blood flow was achieved when doses greater than 50fmole/25~LI/min were infilsed Figure 6A shows changes in arterial blood pressure following ~tlmini~tration of NorLeuYIHPF at 100pmole/25~1Vmin (open circles), 50fmole/25~1l/min (open squares) or saline control (closed squares). Figure 6B shows changes in renal blood flow following a~ Lion of NorLeuYI~F at 100pmole/25~1/min (open circles), 50fmole/25~1/min (open squares) or saline control (closed squares).
FIGURES 7A-7D, 8 and 9 are graphical leprese~ ;ons of AIV binding, as described in Example 6. Figure 7A shows the results of kinetic analyses measuring binding of AIV to coronaly venule endothelial cells (CVEC) showing Ill~illlal equilibrium binding in about 60 mimltes with an appal~;llL Ka of about 9.3 x 107 M-l.
Figure 7B shows the results of kinetic studies measuring the dissociation of AIV from CVEC endothelial cells with an appa,enL Kd Of about 0.3nM. Figure 7C shows the results of equilibrium binding of AIV to 2 separable types of AT4 receptor sites in corollaly venule endothelial cells (CVEC). One type of site with a Kd Of about 1.4+/-0.2nM and a second type of site with a Kd Of about 14.6+/-26.5pM.
Figure 7D shows the results of equilibrium binding of AIV to 2 separable types of AT4 receptor sites in aortic endothelial cells: one type of site with a Kd Of about 4.4 +/- 0.8nM and a second type of site with a Kd Of about 26.9 +/- 9pM. Figure 8 shows c~ ,cLilion of 125I-AIV binding to colonaly venule endothelial cells (CVEC) by non-radiolabeled AIV analogs. Figure 9 shows association of AT2 receptors with G-protein in vascular smooth muscle cells (RVSMC), but non-association of AIV
with G-proteins in endothelial cells (BAEC), as evidenced by the inhibility of GTPyS
to inhibit AIV binding.
FIGURES 10A and 10B show e~-h~nce~ .l of cognitive function, i.e., learning, in AIV intraceleb.uventricularly (icv) injected animals but not in AII-icv-injected animals. Testing of memory was con.i~lcted one day (Figure 10A), or one, two and three days (Figure 10B), after the animals learned a passive avoidance response; as described in Example 7.

WO 94/00492 2 1 3 g 1 0 5 -6- PCr/US93/0603P

FIGURE 11 is a graphical representation of the colllp~Li~/e stability of l25I-AIV (closed dots) and l25I-divalinal (or l25I-VallVal3AIV, open squares) following exposure to rat kidney, as described in Example 4.
FIGURE 12 is a graphical representation of the effects of divalinal AIV (open squares), and divalinal AIV followed by LyslAIV (squares with dots), on blood ples~ule (Figure 12A) and renal blood flow (Figure 12B), as colllpared to saline alone (triangles), saline followed by AIV (closed circles) and saline followed by LyslAIV
(open circles), as described in Example 4.
Detailed Description ofthe Pler~lled Embodiment As used herein the following terms are int~nded to mean the following, namely:
"Angiotensinogen" is used herein to refer to a peptide having the sequence AsplArg2Val3Tyr4Tl~sT-Ti~Gpro7phegHissLeulovalllIlel2Hisl3serl4 abbreviated DRV~ iLVIHS (SEQ. ID. NO. 1) "AI" and "angiotensin I" are terms used to refer to the decapeptide fragment of angiotensin having the N-terminal sequence AsplArg2Val3Tyr4IlesHis6Pro7PhegT-TicgT eu1O, abbreviated DRVYIHPFHL (SEQ. ID. NO. 2).
"des-Asp AI", "d-Asp AI" and "des-Asp angiotensin I" are terms used to refer to an angiotensin polypeptide having the N-terminal sequence ArglVal2Tyr3Ile4His5Pro6Phe7His8Leug~
abbreviated RV~ lL (SEQ. ID. NO. 3).
"AII" and "angiotensin II" are terms used to refer to an angiotensin, e.g., an o~;~apep~ide, having the N-terminal sequence AsplArg2val3Tyr4Ile5His6pro7phe abbreviated DRVYIHPF (SEQ. ID. NO. 4).
"Am," "angiotensin m,~ "Des-Asp A~," and "AII(2 8~" are terrns used to refer to the heplapeplide fragment of angiotensin having the N-terminal sequence ArglVal2Tyr3Ile4His5Pro6Phe7, abbreviated RVYIHPF (SEQ. ID. NO. 5).
"AIV," "angiotensin IV," "A~1~3~)," "Am(2 7~," or "Des-Arg Am" are terms used to refer to the heAapepLide Çl~l"en~ of angiotensin having the N-terminal sequ~once VallTyr2Ile3His4Pro5Phe6, abbreviated VYIHPF (SEQ. ID. NO. 6). In the context of usage herein "AIV" refers to physiological angiotensin II(3 8~ fr~gm~nt~

3 5 formed in a variety of animal species. An "AIV peptide ligand" is a ligand capable of binding to an AT4 receptor. AIV is a ,~plese"La~ e example of an AIV peptide ligand, as are AIV analogs.

vo 94/00492 PCr/US93/06038 "Des-x," also abbreviated "d-x," is used to refer to an amino acid sequence that lacks the amino acid residue "x". Des-Asp AII is used to refer to an angiotensin II lacking the N-terminal Asparagine residue; d-Val(l)AIV is used to refer to AIV
lacking the valine residue (position 1 ) at the N-terrninus of AIV.
S "N-terminal" and "N-terminus" are used interc.h~nge~bly to refer to theNH2-amino terminus of a peptide. The N-terminal amino acid is the amino acid located at the NH2 terminus of the peptide.
"Peptide" and "~cl~,c~lide" are used hl~c~ e~bly to refer to a serial array of amino acids peptide bonded one to another of at least three amino acids in length to plcîclably six amino acids in length, but also up to many hundreds of amino acids in length.
"AIV Ligand" as used herein refers to a compound that is capable of filling the three-dimensional space in a Icceptor binding site so that electrostatic repulsive forces are ...;.~;...;~e~, electrostatic attractive forces are l.,ax;...;7ed, and hydrophobic 15 and hydrogen bonding forces are ~,.;,;~ed Rep,esc,lL~lh~e ligands include "AIV
peptides" and "AIV analogs". T ig~n~ls bind to their specific receptor in a specific saturable manner, e.g., specificity may dctclll hled by the ability of an AIV ligand to bind to an AT4 receptor in a manner that is not co,lll)cLi~ ely inhibited in the presence of an excess (e.g., 1000-fold molar excess) of a co",~ct-lor peptide (e.g., AI or AII).
"AIV peptide" is used interrh~nge~bly with "an~;iGt~-~ IV peptide" to refer to an AIV ligand that is a peptide having, or co--c~l.onding to, at least three of the N-terminal ten amino acid residues (preferably three of the N-terminal eight amino acid residues, and most plcrel~bly three of the N-terminal six amino acid residues), comprising three amino acids selected from among V, Y, I, H, P, F, L, K, A, H, NVal, NLeu, or Orn; prcrcl~bly from among V, Y, I, P, K, NVal or NLeu; and most plcrclably from among V, Y, K, NVal, or NLeu. Replcsenlali~e eAan",les of AIV
peptides have an amino acid seq~l~nce related to the AIV N-terminal sequence VYIHPFX, i.e., by conservative and nonconservative sub~ ul;on~ of amino acids, or by derivatization or covalent modification, (as desc,ibed below), and ~I,e[cin X is any non-interfering amino acid. Re~,t;senl~ re AIV peptides are polypeptides from 3 amino acids in length to many tens of amino acids in length. Other rcplese~llali~/e ~Y~mrles of "AIV peptides" include peptides that are capable of antagoni,i"g binding of "AIV" to its receptor, i.e., "antagonists" (as defined below), and other "AIVligands" are capable of binding to the AT4 receptor and exerting effects similar to "AIV", i.e., "agonists" (as defined below).

WO 94/00492 PCI/US93/060~
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As used herein the term "AIV analog" is inte.n~ed to mean a chemical compound that mimics or improves on the electronic, steric, hydrophobic, and 3-dimensional space-filling requirements of the conctituent amino acid residues involved in binding of the AIV peptide to the AT4 receptor (e.g., a mimetic chemical 5 AIV composition). AIV analogues may be polypeptides, i.e., having amino acids bonded by peptidic linkages, or may be non-peptides, i.e., having amino acids not bonded by peptidic linkages. Representative examples of AIV analogs include chemical mimetic compounds that are capable of antagol i~ing binding of AIV to its receptor, i.e., antagonists (as defined below), and other AIV ligands are capable of 10 binding to the AT4 receptor and exerting effects similar to AIV, i.e., agonists (as defined below).
"Agonist" as used herein means an AIV peptide or AIV analog that is capable of spacially collrul~ l-g to the molecular space filled by an AIV ligand and that is further capable of colllbillillg with AT4 lecel)lol~ to initiate an action that is initi~ted 15 by a physiological AIV molecule when it binds to its specific AT4 receptors on cells in vivo or in vi~ro. Represell~ e examples of actions initi~ted by AIV are illustrated in the Examples. Agonists possess binding affinity for AT4 receptor(s) and intrinsic activity for in~lçing the activities that are in~lced following the binding of AIV to AT4 receptor. Represenlali~e examples of agonists include VYIHPFX, NvaYlHPFX, 20 and OrnYI~FX, wherein "X" is used to decign~te one or more non-hllelr~ling amino acids. Representative examples of processes for recognizing agonists are described in Example 4.
"Antagonist" as used herein means an agent that spacially conforms to the molecular space filled by an AIV ligand and that is further capable of colllbinillg with 25 the subject AT4 rece~,lol(s) to inhibit, neutralize, impede or reverse, at least in part, an action of physiological AIV when it binds to its specific AT4 receptors on cells.
Repleselllali~re examples of antagonists include KYI~FX, and NLeuYI~FX, whelein "X" is used to deign~te one or more non-inle,Ç~,i"g arnino acids.
Representative examples of processes for recognizing antagonists are described in 30 Example 4.
"AII ligand" as used herein refers to a peptide having the N-terminal amino acid sequence DRVYI~FX and capable of binding to an AT1 or AT2 AII receptor, where X is any non-intelre,ing amino acid.
"Non-interfering amino acid" as used herein means any amino acid that 3 5 when introduced into the C-terminus of an AIV peptide ligand does not interfere with binding of the AIV peptide ligand to its specific AT4 receptor.

~0 94/00492 21 39 1 05 PCI/US93/06038 ._ , "ATl" and "ATl recc~Jlorll and are terms used interch~ngeA~kly to refer to a receptor subtype capable of binding AII.
"AT2" and "AT2 rcc~,lor" are terms used interchangeably to refer to a second receptor subtype capable of binding AII.
"AT4 f~c~;~.lor"is the term used to refer to a receptor capable of binding an AIV ligand but not an AI, AII, or AIII ligand.
"AT4 rec~,lor fragments" is a term used herein to refer to portions of the AT4 receptor that are smaller in size than an AT4 receplor isolated from a natural source, e.g., tissues, biological fluids and the like, but remain capable of binding AIV.
FragmPnts may be pley~ed from an AT4 receptor isolated from a tissue and then subjected to proteolytic degradation or trç~tmPnt with a ~.hPmic.~l such as cyanogen bromide. In the latter case the rli~g.~ s of the receplor are conveniently purified before use, e.g., by reverse-phase HPLC or immllne affinity chlol"alography.
Alternatively, fr~gmP.nts of the AT4 l~ceptor may be yley~ed by eAyiession of a portion of a nucleotide sequence of a genomic or cDNA clone capable of eAylessing the AT4 receptor, e.g., a portion of the AT4 nucleotide sequence in an c~ylt;ision plasmid or vector introduced into a cell, wl,e~ei" the cell m~mlf~ctllres the AT4 receptor fragment and the fragment can be purified (as above). For example, fr~gmPnt~ of the AT4 receptor that contain the AIV ligand binding domain of the receptor may be soluble in biological fluids and aqueous solutions and may bind AIV
ligand with a greater or less Kd than AT4 receptor under these conditions. The binding affinity",Ayl ~ssed as the Kd, of the AT4 receptor fragment for an AIV ligand is about 30nM to about 0.003nM, plerel~bly about lnM to about 0.01nM, and most preferably the binding affinity is about 0.5nM to about 0.01nM.
"Triggering the AT4 ~cce~,lor," "acti.ali,.~ the AT4 fece~tor," or "activation of the AT4 r~C~;IJtOr" are used interch~ng~hly to refer to col~ll"aLional and/or structural or activity ~h~l~ges resident in an AT4 receptor following binding of an AIV ligand; e.g., col~""~Lional rhAr~es may be evident by cl,anges in the near W spectra of the receplor or ch~nges in the circular dich,ois", (CD) spectra;
structural ~ h~nges may be evident as covalent modification of the receptor, e.g., by phosphorylation; and, activity c.l-~np,es may be evident as an increase in enzyme activity, e.g., an innate tyrosine kinase activity. A receptor that has interacted with an AIV ligand and has undergone the process of "trigge~ing" is also referred to herein as a "triggered AT4 receptor. "
"Substantially purified" as used herein refers to a prel)al~lion that colllaills a peptide, ligand, or receptor that is enriched greater than about 10-fold from the WO 94/00492 PCr/US93/0603Y
~ Z139i~5 -10-natural source material, e.g., membrane plepal~ions of a tissue, and that also contains less than 5% impurities detect~ble by one-dimensional SDS-PAGE. The subst~nti~lly purified AT4 receptor approaches homogeneity at purification levels greater thanabout lOOOx.
The term "AIV angiot~ r~ e" as used herein refers to a dipeptidylpeptidase capable of catalyzing hydrolysis of an arginine-valine peptide bond in an angiotensin, e.g., AI, AII, or AIII, without catalyzing hydrolysis of any of the other peptide bonds in the angiotensin.
"Pressor activity" is used to refer to blood pressure changes ind~lced by an agent, e.g., AII.
As diccncsed above, the angiotensin field has often been fraught with complexity and conflictinE h~rul.l.alion. During attempts to purify the angiotensin II
(AT2) receptor from bovine adrenal cortex, the curious observation was made that as purification proceeded the appa~en~ spe~ifirity of the receptor ~.h~nged While isolated ~--clll~ es bound stable AII analogs better than AIII, the solubilized receptor c~ ed the opposite order of ligand specificity with AIII binding betterthan AII. Following purification it became app~rc..L that the receptor had all but lost its ability to bind AII, and was slowly losing its ability to bind Am, despite taking steps to inhibit proteases. Previously it had been reasoned that 10% hydrolysis of 20 ligand was h~conse4~1ential; however, considering the strange behavior of the receptor during purification, the possibility was considered that a portion of the confounding data could be explained by metabolic conversion of the active AII and AIII ligands to previously l-nrliccP.rned metabolite(s) and interaction of the metabolite(s) with a novel receptor(s). The N-terminus of both AII and AIII are reportedly labile to proteolytic 25 hydrolysis, but previous studies did not s~ticf~ctorily control for such hydrolysis.
Thus, the possibility was considered that previous studies may have been confounded by the conversion of one ligand (i.e., AI or AII) into another (i.e., Am or AIV), e.g., medi~ted by renin, angiotensin co..~c l;ng enzyme (ACE), ~.i..opepLidases, endopeptidases, and/or c~l,u~yl,c~,Lidases (e.g., through reactions such as those 30 depicted in Figure 1). Considering the previous studies in this light, physiological activities reportedly triggered by the AII or AIII peptides, on rc-P-~,.,;n,.l;Qn, seemed to require higher levels of peptides than would be predicted from the physical binding propc.Lies of the rcspecLi~lre AT1 or AT2 receptors. However, if a previously unrecognized receptor existed it might be reasoned that the relatively high levels of 35 AII or AIII are required to provide precursors for conversion to metabolites that would bind to the novel receptor. To approach this problem an improved assay ' ~ 94/00492 ~ 2 1 3 9 1 0 S PCl/US93/06038 -1.1-system was developed. After an exhaustive search, a rapid spin chromatographic technique was developed to separate bound from free ligand, and an assay buffer was discovered that ...;~ ed N-terminal degradation of angiotensins. In addition, a stable N-substituted analog of AII was used as a control (i.e., Sarl,Ile8-AII), so that S classic AII binding sites (20) could be identified. Under these conditions, the possible conversion of AII to AIII or other metabolites is klimin~tefl, and AII binding sites are accurately idçntified When assays were con-J~cted under these conditions to determine what angiotensin fr~gmknts bound to bovine adrenal cells, it was surprisingly observed that the 125I-AII or 125I-AII radiolabel "specifically bound" to 10 the cells was a hexapeptide fragment of AIII consisting of residues 3 through 8, i.e., AII(3 8~. Further, it was discovered that the amount of binding in a given AII (or Am) prcep&l~lion was directly proportional to the amount of the hexapeptide in the prel)~alion The use of 125I-radiolabeled h~,~?cl~Lide as the ligand in the receptor assay dramatically increased binding. A reevaluation of binding in purified Illtlll~ e 15 plepal~lions demonstrated the presence of two di~erclll and distinct receptors, one for AII and a second for AII(3 8~. Further, neither AII nor AII(3 8~ ligand effectively displaced the other. The results, thus, SLl ol1gly suggested the presence of two distinct receptors, one for AII and a second for AII(3 8~. HereinarLer AII(3 8~ is lerclled to as AIV and the novel AII(3 8) cceptor is lercllcd to as the AT4 receptor. The notion of 20 two separate and distinct receptors was cor~,lled by solubilizing, isolating, and subst~nti~lly purifying the AT4 receptor under conditions that did not solubilize the AT2 receptor.
The experimental results described in the Examples, demonstrate for the first time the ~xi~tknce of a distinct high-affinity cellular receptor that specifically binds the 25 h.,A~pcl~Lide fragment of angiotensin AII, i.e., AII(3 8~, termed herein angiotensin IV, or simply AIV. The angiotensin AT4 receptor is characterized in the Ex~mples, with respect to structural IC~IUilClllCllL~ for ligand binding, species and tissue distribution of the receptor, physiological role of the AIV ligand-AT4 receptor system, intracell~ r Illk~sçl~g~l ~ign~lin~ pathways activated by the receptor, conditions for isolation and 30 purification, and molecular size of the recel)tor.
In one embodiment of the invention, compositions are provided which comprise subst~nti~lly purified angiotensin AT4 recep~or or rl;.~ thereof, that are capable of binding an angiotensin AIV ligand but not an angiotensin AI or AII
ligand. The AT4 receptor binds AIV ligands, and does not bind to a peptide having 35 the AII N-terminal sequence, i.e., DRVYI~F. AT4 receptors of the invention are specific for AIV and AIV ligands, and are more fully characterized by the following WO 94/00492 ~ Q~ -12- PCr/US93/0603P

properties a) AT4 receptor has a Kd for AIV of about 30nM to about 0 003nM, preferably about 3nM to about O OlnM, and most preferably about lnM to about O lnM (representative examples of binding p-opellies of AT4 receptors are summarized in Table 1); b) AT4 receptor binds to AIV ligands in a saturable and 5 reversible manner; c) the binding of an AIV ligand to the AT4 receptor is compe~ ely inhibited less than about 1% to about 10% by an angiotensin AII
prep~lion (e g, Sarl,Ile8-AII) that contains less than 0 1% of an AIV ligand when the competition of AIV binding is measured in the presence of about a 1000-fold molar excess concentration of the co...pe~ing ligand using the assay conditions 10 described in Example 1 In a represe..L~Li~e embodiment, AT4 receptors having these plopellies may be isolated from bovine adrenal cortical ...e...~ es (e g, described in Example 1) Isolated AT4 receplo-~ from this source have the kinetic, equilibrium binding, and physical propcl Lies set forth below in F~mple 1 The AT4 receptor of the invention has a molecular size of about 120kD to about 200kD on SDS-PAGE, preferably about 140kD to about 160kD, and most preferably about 140kD to about 150kD
For example, an AT4 receptor of the invention is present in ll~u~b-~ne p- ep~ alions of adrenal glands of most ~ n species (e g, cow, pig, horse, dog, cat, rabbit, and guinea pig) and, as purified from bovine adrenal ~c~nb~1es, the AT4 receptor has an 20 appale-.L molecular size of about 146kDa on SDS-PAGE AT4 receptors are also cA~ressed in guinea pig aorta, heart, kidney, liver, lung, vascular smooth muscle, pituitary, and uterus, as well as vascular endothelial cells and brain ''~94/00492 ~1i3~S PCI/US93/06038 Bindin~s Plope~ lies of AIV Receptors Animal T ssue Ple~ lion K~l nM) Bn~ ~xC Example #
Rabbit .-:eart ~ ~ f.C overal: 1.70 731 i~2 Guineapig .-.eart ~.. , .. i.i-~C site#: 1.33 144 i~l Bovine A~renal ~ . .. ~ - c 0.54 1030 i~l Cortex Sol. Receptor~ 0.51 87.9 #1 Adrenal~ ' -- -- 397.3 #2 Medulla Bovine HeanVasc. Endo.e overall: 0.7476 ~2 site#l: 26.5 6 i~7 site#2: 1.4 594 i~
Bovine HeartAorticEndo.~ site#l:26.910 ~~
site#2: 4.4 434 ;~
Guinea Pig Brain I-rr ~ ~ 0.1 306 #11 g HSTAh 0.11 ~l~ j Cerebellum 0.2''.3'' ~::
Brain Stem 0.9 :~
GuineaPig ~orta .:.eart '~ y_ ~dney " -- ''2.7 i~
_iver " -- "8.9 i~~
.,ung ~-.erus " -- 7 i~2 Pig ALrena ~.. ~.,.. ~c ~ 3~7.3 i~2 Horse ALrena " -- -0.8 ~2 Dog ALrena " -- -2.7 i~2 Cat ALrena " --- 199.6 ~2 Rabbh ALrena " -- 105.3 i~2 Guinea Pig ALrena " - 101.2 i~2 c.) BmaX= ~ 1 b nding under equilibrium b nding concitions, (fmol/mg protein), d.) sol. rec~lo,- solubilized receplo~, e.) vasc. endo.= vascular endothelial cells CVEC;
) aortic endo.= aortic endothelial cells BAEC;
g.) hippocampus= hippocampal solubilized receptor; and, h.) HSTA= hypoth~l~mllc~ th~l~ml~c, septum, a.,Leleo~entral third ventricular areaofbrain.
- The invention further provides AT4 receptor ligands that specifically bind to, activate and/or antagonize the AT4 receptor. The AIV ligands generally comprise at - least 3 of the N-terminal amino acid residues of AIV, or analogues or AT4 receptor binding equivalents thereof. The amino acid residues of the ligands may be bonded by WO 94/00492 ` ~ ~j`~ PCT/US93/0603 peptidic linkages, or may be bonded by non-peptidic linkages. The ligands generally have a Kd for the AT4 receptor below about 3 x 1 O~M.
Generally, the AIV ligands of the invention are based on the structure of AIV.
The AIV ligands may be obtained by constructing AIV analogs that have one amino 5 acid substituted for by another of like plop~lLies, i.e., a neutral polar amino acid for another neutral polar (e.g., G, A, V,I,L,F,P, or M), a neutral nonpolar amino acid for another neutral nonpolar (e.g., S, T, Y, W, N, Q, C), an acidic amino acid for another acidic (e.g., D or E), or a basic for a another basic (e.g., K, R, or H). The AIV ligands may alternatively be obtained by constructing an AIV analog that is 10 covalently modified, e.g., wherein an amino acid residue is substituted by amidation, adenylation, methylation, acylation, phosphorylation, uridylation, fatty-acylation, glycosylation, and the like to form a "s~sliluled amino acid residue". In addition, the AIV ligands of the invention may contain one or more stereoisomers of the cor..~ Pnt amino acids residues; i.e., may contain one or more substituted or unsubstituted amino acid residues in the D-configuration.
In other embodimpntc~ the invention provides angiotensin AIV ligands and ligand compositions that include AIV analogs, AIV peptide derivatives, and covalently modified AIV peptides, all of which are capable of binding to an angiotensin AT4 receptor. AIV ligands of the invention are generally defined by the formula Rl-R2-R3-X
wherein Rl is a substituted or ullsubs~ ted amino acid residue having a neutral or positively ch~;ed ~liph~tic side chain Zl, said amino acid being selected from among V,I,L,A, G, F,P, M, K, norvaline, norleucine, and ornithine;
R2 is a sub~liluled or unsubstituted neutral nonpolar amino acid selected from the group concicting of Y, W, N, Q, F, or C;
R3 is a substituted or unsubstituted neutral polar amino acid sPlected from the group co~ g of G, A,V,I,L,F,P, or M; and X is nothing, R4, R4-R5, or R4-R5-R6, wherein R4 is a substituted or unsubstituted basic amino acid residue sPIected from the group colls;sli,lg of K, R and H, R5 is a substituted or unsubstituted neutral polar amino acid residue selected from - the group concicting of G, A, V,I, L, F, P, and M, and R6 is a substituted or unsubstituted neutral polar amino acid residue sPlected from the group Col,S.sli~lg of G, A, V,I, L,F, P, M, and polyamino acid residues co..l~it~ g one or amino acid 3 5 residues which do not prevent binding of the AIV ligand with the AT4 receptor.

-'~94/00492 7~ 3910S PCI/US93/06038 Thus, the AIV ligands of the invention are generally amino acid chains that contain 3, 4, 5, or 6 amino acid residues corresponding to the N-terminal 3, 4, 5 or 6 amino acid residues of AIV (the polypeptide, VYIHPF), or may optionally extendedat the C-terminal end with one or more amino acid residues that do not prevent 5 binding, due to spatial, co~ alional, electrostatic or other considerations, to the AT4 receptor. The amino acid residues may be linked in the amino acid chain by peptidic linkages to form peptides, or the AIV ligands of the invention may contain one or more non-peptidic linlf~g~c, such as methylene or C-N linkages, to enhance metabolic stability or other properties of the AIV ligands, as is hereinafter further 10 described. Rep.t;se~ /e AIV ligands of the invention include, but are not limited to C-terminal trunc~ted forms of AIV, such as AIV(1 5), AIV(l 4), and AIV(1 3);
stereoisomerically modified forms of AIV, such as D-H4 AIV, D-P5 AIV, and D-F6 AIV; full or trlmc~ted forms of AIV with modified amino acid re~i~luee7 such as G4 AIV, G5 AIV, G6 AIV, Nlel AIV, Kl AIV, F AIV, Il AIV, Pl AIV, Nval AIV, Ornl 15 AIV, Y6 AIV, I6 AIV, NleYI, KYI, and MeYI, derivatives of AIV with one or more non-peptide linkages belween amino acid rç~iduçc, such as Me all AIV (whereill the dçeign~tion all refers to a methylene -CH2- linkage belween the amino acid residue in position 1 (Nle) and the amino acid residue in position 2 (Y)), Me all Val3 AIV, Kall Val3 AIV, Kall AIV, Vall AIV, Val3 AIV, and Vall Val3 AIV, and substitued AIV
20 ligands, such as propal1oyl-N oml AIV, O-me Y2 AIV, isobutyl-N oml AIV, N-me Il AIV, NleYI amide, KYI amide, MeYMe amide, MeYNva amide, Me al N-me YI
amide, benzyl Cl AIV and the like.
The physical plope-lies of the AT4 receptol~ that dt;lelll~ e binding of the AIV ligands were mapped using synthetic peptides and analogs, as described below in 25 detail in the e,.~nplEs. The structure of the N-temlinus of AIV is most important for high affinity binding of an AIV peptide to an AT4 receptor. The AT4 receptor binding site is a cool.li..aled multidomain binding site ~hereil1 binding in onesubdomain may be ~y~lllded by high affinity binding at a second subdomain through an intluced co,~ulmalion change in the AT4 receptor binding site l-ydlophobic pocket 30 subdomain. At least three binding site subdom~in~ in the AT4 receptor were .napped using syllllletic peptides and analogs. The binding site is stereospecific at a first subdomain for L-Valine in N-temlinal amino acid position 1 (Vall) of AIV; at a second subdomain for L-Tyrosine in position 2 (Tyr2) of AIV; and at a third site for L-isoleucine (Ile3) in position 3 in AIV. The results suggest that Vall in AIV may 35 interact laterally with the walls of the groove of the receptor while Tyr2 in AIV may interact with the receptor binding site through van der Waals forces and hydrogen WO 94/00492 PCr/US93/0603 213gl0S -16-bonding. AIV peptides having a weak hydrophobic amino acid at the N-terminus with an aliphatic side chain (e.g., KYIHPF, NleYIHPF, OrnYIHPF) bind to the AT4 receptor with a higher binding affinity than AIV (binding of KYIHPF is 50-fold higher than AIV, and NleYIHPF has a Ki Of about 1O-l2M). N-terminal extension of AIV is5 incompatible with binding, as is deletion of the N-terminal valine (Vall) residue.
Deletion of Vall reduced binding affinities 1000-fold; substitution of Vall with Sar decreased binding affinity; addition of D-arginine to the N-terminal Vall reduced affinity for the receptor by 100-fold. The receptor binding site domain of the AT4 receptor COllL~llS a hydrophobic pocket COl~llllillg closely to the space filled by 10 norleucine (i.e., çng~ging the Vall residue of AIV) and in close apposition with a negatively chalged residue (i.e., çnp~ging the primary amine of the N-terminus of Vall). Removal ofthe N-terminal amino group decreases by 1000-fold.
The C-terminus of the AIV peptide is relatively less important in the receptor binding and C-terminal extension of AIV ligands of the invention with "X" is allowed.
15 However, removal of both the Pro5 and Phe6 residues from AIV reduced binding affinity by about 21-fold to a Ki Of 500nM. The C-terminus of the AIV peptide may determine receptor subtype specificity of binding.
In addition, it has been found that AT4 receptors isolated from bovine adrenal cortical ",t".l"~nes do not effectively bind AIV peptides synth~ ed with an 20 N-terminal extension with Sar or GABA. Nor do the illustrative AT4 receptors effectively bind peptides having the N-terminal L-Val replaced with D-Val or Sar.
Also, removal of the N-terminal L-Val from AIV all but elimin~tes binding to the AT4 receptor. AT4 receptors of the invention have a receptor binding site that is stereospecific for L-Valine. In one illustrative example, D-VallYIHPF has 1000-fold 25 lower binding affinity for the AT4 rece~or than L-VallYI~F. The illustrative AT4 receptor icol~ted from bovine adrenal cortical ",e",~ es coll~ains a binding site that prefers weak hydrophobic amino acids in the number 1 position (i.e., Rl) of the AIV
ligand, i.e., h.cleas,.,g hydlophobicity by repl~ing Vall with Phe (i.e., FlYIHPF) decleases binding affinity 4-fold, but repl~cPmlont of Vall with another weak 30 hydrophobic amino acid (i.e., IlYIHPF) results in only a slight change (an increase) in binding aff~nity. For high affinity binding of an AIV peptide to an AT4 receptor the structure of the N-terminal neutral polar amino acid is most i...~,o"~. N-terminal extension is incol..pa~ible with binding, deletion of the terminal valine residue ";~ es binding (Ki >10~), substitution with Sar decreased binding affinity, 35 substitution with Ile results in equivalent binding, substitution with Phe resulted in a 5-10-fold decrease in the affinity of binding, Pro-substituted AIV peptides bind with 21391o~
~~ ~ 94/00492 PCr/US93/06038 100-fold lower affinity, Lys-substituted AIV peptides bind with 10-fold higher affinity, and AIV ligands having a norleucine in the number 1 position (also abbreviated herein Me, NLe, NLeu, NLeul, or Mel) bound with 1000-fold higher affinity.
The interaction belween the AT4 receptor binding site and AIV ligand may be dictated by requilelllellls for an AIV ligand co"l~ ;"g a flexible aliphatic carbon side chain, (i.e., as opposed to a relatively rigid aromatic ring), rather than by the degree of hydrophobicity of the side chain. In a representative example, substitution of Vall with Aspl (i.e., to form AlYIHPF) results in an analog with no binding affinity for the AT4 receptor (i.e., has a Kd > lO~M). Further, the AT4 receptor binding sites of the invention may prefer a flexible aliphatic carbon side chain having 4 carbon atoms that lack a positively charged residue. Heptanoyll AIV with a 5 carbon side chain hasreduced affinity as colll?ared to Mel AIV. In a represellla~ e example, MelYIHPFhas higher binding affinity for an illustrative AT4 receptor than LyslYIHPF, which was higher than NVallYIHPF, which is in turn higher than OrnlYIHPF. The AIV
peptide ligands of the invention having norleucine substituted for Vall (i.e., MelYIHPFX) are partial agonists of VYIHPFX binding to the subject AT4 receptor and have an app~t;llL Ki Of about 1 x 10-12M.
The AT4 receptor binding site interacts specifically with the N-terminal amino acid residue (i.e., Rl ), and the latter interaction is specific with respect to both absolute space occ~p~ncy volume (i.e., of the receptor binding site) and charge (i.e., of the AIV ligand). In representative examples, methylation of isoleucine in Ilel of IlYIHPF (i.e., to form CH3-IlYIHPF) reduces affinity of the illustrative receptor for the peptide by 67-fold; substitution ofthe Vall primary amine (NH3) with a secondary amine (-NH-; in this case by sllbstitllting Prol for Vall, to form PYIHPF) reduces the affinity of binding to the illustrative receptor by 8-fold; substitution of Vall with benzoic acid or 6-amino-hexanoic acid gives peptides with a Kj>lmM; and, replacing Vall with GABA (gamma-amino butyric acid; to form GABA-YIHPF) decreases binding affinity by 250-fold for the illustrative receptor.
The AT4 rec~lor binding sites of the invention also appear to be stereospecific for Tyr2 (i.e., Y) in the R2 position of the subject AIV peptide ligands.
In lt;presell~ e examples, substitution of D-Tyr2 or Phe2 (with a benzyl ring) for Tyr2 (with a phenolic ring) results in analogs (i.e., V[D-Y2]IHPF, or VF2IHPF, respectively) with very low affinity for the illustrative adrenal cortical receptor.
Phenolic side chains in the Tyr2 residue may also interact with residues in the subject AT4 receptors through hydrophobic and/or hydrogen-bonding.

WO 94/00492 ~39 -18- PCI/US93/060.~"

The AT4 receptor binding sites of the invention tolerate repl~rçm~nt of the Vl-Y2 peptide bond with a non-carbonyl bond that has a similar bond length, but is non-planar and has a non-rigid carbon-nitrogen bond. The latter repl~c~mP.nt bond may preferably be lesis~ to proteolytic hydrolysis thereby cGl~lling additional 5 stability on the AIV ligand and enh~n~ing utility in therapeutic compositions for oral delivery. In a represelllali~/e example, repl~c~m~nt of the Vl-Y2 peptide bond with a methylene bond reduces receptor binding affinity by only 5-fold; and, repl~cem~nt of both the Vl-Y2 and I3-H4 peptide bonds with methylene bonds results in N-Vl-CH2-NH-Y2V3-CH2-NH-H4P5F6-C (also rerelled to herein as Vall Val3 AIV
10 or divalinal AIV) that has an affinity equal to or better than VYIHPF.
The binding site of the AT4 receptors of the invention is a coor(lin~te~7 mlllti-lom~in binding site wht;lt;ill binding in one subdomain ofthe binding site may be enhanced or inhibited by binding at a distant second subdomain. In one representative example, substitution of Ile for Phe at the R6 position of VYIHPF6 results in an15 analog (i.e., VYIHPI6) that binds to AT4 receptor (i.e., through the Vl subdomain sites) with a higher affinity than the parent VYIHPF molecule. In a second representative example, substitution of Ile6 for Phe6 in KYIHPF6 results in an analog (i.e., KYIHPI6) that binds to the receptor (i.e., through the Vl subdomain site) with a lower affinity than the parent KYIHPF6 molecule. The C-terminus of the subject AIV
20 peptide ligands appears to be relatively less important in receptor binding. In replt;selllali~e examples disclosed below, deletion of the C-terminal Phe6 from VYIHPF (i.e., to form VlY2I3H4P5) does not alter binding ~ignific~ntly; C-terminal extension with hi~ti~ine does not alter binding (i.e., to form VlY2I3H4P5F6H7); and, addition of both his and leu reduces affinity only 2-fold (i.e., VlY2I3H4P5F6H7L8).
25 Truncation of the C-terminus, i.e., at the R5 position decreases binding. In a replesenlali~e example removal of Pro5 from VYIHP to give VYIH, decreases binding 21-fold, and gives an analog with a Kj>500nM. The binding site domains of the subject AT4 receptor of the invention recognize the N-terminus of the subject AIV peptide ligands with a high degree of specificity and while the receptor interacts 30 less closely with the C-terminus this region of the subject AIV ligand may determine recel,lor subtype specificity.
In another embodiment of the invention, antagol~sls of AIV are provided that bind to the AT4 receptor. Plesenlly particularly plerelled antagonists of the invention include the non-peptide divalinal AIV and the C-terminal substituted tripeptide MeYi 35 amide, as described in Example 4, although other antagonists will be readily appare from the data and disclosure set forth herein.

. .

~ 94/00492 2 ~ 3 9 1 0~: PCr/US93/06038 Other aspects of the invention include processes for identifying AIV peptide ligands, i.e., by structural ~ ;on of the receptor binding requirements of test prepa,~Lions (e.g., with respect to both blocking and/or promoting binding of the alternative peptide) to AT4 receptors such as those in heat-treated purified membrane 5 preparation that are free of peptidase activity and devoid of other angiotensin receptors, i.e., AT1 or AT2 receptors. (Examples of such heat-treated membrane plep~Lions and assay methods are provided in the examples, below.) Those skilledin the art will recognize that the binding activity of any AIV peptide can be tested, e.g., using the receptor binding assays described herein, and that analogs, AIV peptide derivatives, and covalently modified AIV peptide or non-peptide ligands may exhibit activity as antagonists, agonists, promoters, or enhancers of AIV binding to its AT4 leceplor. C~n-lid~te AIV peptides may be prepared with substitution of other L-amino acids having di~relll steric, electronic, and hydrophobic character for the L-Val in the natural AIV ligand. Skilled artisans will also recognize that a similar approach may be used to characterize further the role of C-terminal amino acid residues in binding of a peptide to the AT4 receptor, (i.e., other than the C-terminal P
and F). Substitutions and modifications of internal amino acids (i.e., Y, I, or H) can also be c~ ed by constructing the approp,iate series of D-substituted, covalently modified, derivatized, or deleted peptides. The first or second meSse~er intracçlh.l~r pa~h~lvays triggered in cells by interaction of an AIV ligand with an AT4 receptor may be used to test a series of peptides, analogs, derivatives, or covalently modified AIV
peptides for their ability to bind to the AT4 receptor and trigger the intraCçl~ r signal. For in.~t~nce7 activities such as tyrosine kinase, guanylate cyclase, Protein kinase C, Ca~ flux Gh~ges7 phospholipase C (PLC) activity, or prost~gl~n~lin or endocrine or exocrine hormone release from cells, may be monitored to determine whether the peptide triggered the AT4 receptor, and the receptor then signaled an increased or decreased activity in the cell.
In all cases, the AIV peptides, AIV analogs, agonists and antagonists, and derivatives and covalently modified forms of the AIV peptides of the invention are recognized by their ability to bind the AT4 receptor with an equilibrium dissociation cons~ (Kd) below 3 x 10~M, more preÇt;,~bly below 3 x 10-8M and most ple~lably below 3 x 10-9M, and to a low binding affinity for AT1 and AT2 recep~o,~
with a Kd greater than 1 x 10~M.
In still other embodiments of the invention, processes are provided for identifying and characterizing a physiological effect of an angiotensin AIV peptide by assaying the effect(s) of the peptide on a selected in vitro cellular process. For 2~ 39~0S -20- PCI/US93/0603~

imt~nce, to identify and characterize the physiological effects of an AIV peptide on blood flow, it may be convenient to assay renal blood flow, or in vitro cellularprocesses of endothelial cells and/or vascular smooth muscle cells. To identify and characterize a physiological effect of an AIV peptide on cardiac ventricular hypertrophy, assays may eY~mine the effects of an AIV peptide on growth of a cardiomyocytes in vitro. The processes disclosed herein are also useful in identifying how the in vitro activities of physiological AIV may be blocked or promoted by AIV
peptides, AIV analogs, or derivatives or covalently modified forms of AIV peptides, as well as AT4 receptor fr~gmPnts and the like. Representative examples of useful assays for identifying the subject AIV peptide ligands and AIV ligands are provided in the C;A~n~
As used herein the term "cellular processes" is inten~led to mean biological activities that may be measured in vitro or in vivo by q~ e and/or q~ it~tive assay. For example, cell growth or metabolism may be measured (e.g., radiolabeled amino acid synthesis into protein, glycolytic activity, oncogene cAI,ression, and the like); or, proliferation (e.g., 3H-thymidine synthesis into DNA); or, marker eAI.less;on (e.g., }nRNA by Northern, protein by Western blot, antigen by immlmo~ss~y~ in vitro selectable drug-re.cist~n~e marker by cell survival in toxic drug, and the like); or, electrical activity (e.g., in neural cells).
Other aspects of the invention provide compositions and methods for promoting or inhibiting cellular activity of neural cells, e.g., neural motor, cognitive or analgesic activity of neural cells in the brain. The effect of the AIV compounds on motor activity may be observed by e~ alterations in activity as measured with open-field techniques. The cognitive activity may be observed by passive avoidance testing, Morris :iWilllllling maze pe~ro~ ce, and various operant tasks. To assay the effects of an AIV composition on a cellular process it may be useful, for example, to measure cellular processes before and after addition of AIV peptides to make colll,~ e observations in parallel cell cultures. In this manner antagonists, agonists, inhibitors, promoters, enh~ncPrs~ and the like may be identified and characterized with respect to their physiological effects in vi~ro and possible effects in vivo.
When used for therapeutic purposes, the route of delivery of the AIV ligands, AT4 receptor, AT4 receptor fr~gmrntc7 and AIV monoclonal antibodies of the invention is determined by the disease and site where ~ is required. For 3 5 example, the compounds or compositions of the invention may be applied topically, or by intravenous, intraperitoneal, intr~m~sc~ r, subcutaneous, intranasal and ) 94/00492 2 1 3 9 1 0 S PCI/US93/06038 intradermal injection, as well as by intrabronchial inctill~tion (e.g., with a nebulizer), transdermal delivery (e.g., with a lipid-soluble carrier and skin patch), gastrointestin~l delivery (e.g., with a capsule or tablet), intracelebroventricularly (icv) into brain, or intraspinally into cerebrospinal fluid (CSF).
5 The prere"ed therapeutic compositions will vary with the clinical indication.
Some variation in dosage will neces~rily occur depending on the condition of thepatient being treated, and the physician will, in any event, determine the applopliate dose for the individual patient. The effective amount of AIV ligand per unit dose depends, arnong other things, on the particular ligand employed, on the body weight and the chosen inoculation legh"e~l. A unit dose of ligand refers to the weight of ligand without the weight of carrier, when a carrier is used. An effective tre~tm~nt will be achieved in the microenvil~,lllllell~ of the cells at a tissue site as the concentration of AIV ligand approaches a collcellllalion of 10-sM to 10-1lM. Since the pharmacokinetics and pharmacodynamics of these agents will vary in di~lenl species and di~lelll p~tiçnt~, the most prer~lled method to achieve the therapeutic concentration is to gradually esc~l~te the dosage and monitor both the biological effects and the concellL~lion in the biological fluids (e.g., through the use of a diagnostic imm~-no~s~y, or radioisotopic or chemical label). The initial dose, for such an esc~l~ting dosage regilllell of therapy, will depend upon the route of a~mini~tration~ For intravenous a~lmini~tration, for an agent with an appro~illlale molecular weight of 10,000 d~ltone, an initial dosage of appro~hllately 70mg/kg body weight is ~tlmini~t~red and the dosage is esc~l~ted at 10-fold increases in concentration for each interval ofthe esc~l~ting dosage l~hllell. Therapeutic efficacy in this example is achieved at 0.7-70mg/kg body weight of the theoretical 10,000 dalton peptide.
The compounds may be ~d~ el t;d alone or in co,l-billa~ion with ph~.,~celti.~.~lly acceptable carriers, in either single or multiple doses. Suitable pharm~ce~ltical carriers include inert solid diluents or fillers, sterile aqueous solutions, and various nontoxic organic solvents. The pharm~ce~ltic~l compositions formed by colllbi~ g the AIV ligands or receptor f~gmf~nts ~ith the pharm~ce~ltic~lly acceptable carrier are then readily a~mini~t~red in a variety of dosage forms such as tablets, l07f'~eS7 syrups, injectable solutions, and the like. These pharm~ceutic~l carriers can, if desired, contain additional ingredients such as flavorings, binders, excipients, sweet~ning or flavoring agents, colored matter or dyes, emulsifying or suspending agents, and/or. For palell~el~ on~ solutions ofthe AIV ligand WO 94/00492 . ~, PCr/US93/060^
2~ 39~~ -22-or receptor fragment in sesame or peanut oil or in aqueous propylene glycol may be employed.
The present invention further provides processes for isolating inhibitors of an AT4 receptor-AIV ligand interaction by: a) selecting a cell type that expresses the 5 AT4 receptor; b) adding an AIV ligand to a control culture of said cells; c) adding the AIV ligand and a putative inhibitor to a second test culture of the cells; and d) measuring the level of binding of the AIV peptide to the cells in said second test and control cultures. In the case that an inhibitor is present in the plepal~lion, the level of binding in the test culture is lower than that in the control culture. (An 10 example of such a process is provided in Example 1). Those skilled in the art will recognize that this process may be used to identify an inhibitor of an AT4 receptor-AIV ligand interaction in ch r olllaLographic fractions and the like during solubilization, isolation, and purification of said inhibitors, and that the subject inhibitors may act as agonists or antagonists of the action of AIV inril1ced when AIV binds to its specific 15 AT4 receptor.
In other embodiments the invention provides an AIV angiontçn.cin~ce enzyme capable of hydrolyzing a peptide bond bt;lween an al~;lline and a valine residue in an angiotensin polypeptide, e.g., a polypeptide with a DR\VYIHPF N-terminal sequence, wherein "\" indicates the proteolytic cleavage site that gives rise to an AIV peptide, 20 i.e., with an N-terminal seql1çnce related (as described above) to the amino acid sequence VYlHPF. Isolation and s~s~ ial purification of AIV angiotçncin~ce may be conveniently accomplished, for example, by prepalillg an affinity resin having a non-cleavable or slowly-cleavable AIV ligand covalently bound to the resin, e.g., chemically modified derivatives of a peptide in an amino acid sequence selected from 25 among DRVYIHPF, DRVYIHP, DRVYIH, DRVYI, DRVY, DRV, RVY, or NRVYIHPF, NRVYI~, NRVYLH, NRVYI, NRVY, NRV. Operationally, the peptide useful in this assay is selected based on its ability to bind the AIV
angiotç~ ce and to be l~;si;~L~III to cleavage by the enzyrne. A test prep~ ion of a cellular or tissue extract (or a biological fluid sample) is next ch~ llalographed 30 through the affinity resin; the bound polypeptide(s) is eluted, e.g., at low pH and high salt (e.g., pH2-3, and 2-3M NaCl, and the like), or the bound polypeptide is eluted by adding an excess of AIV ligand. The presence of the AIV angiot~ .s;i~ce in the eluate can be dt;lellll,led by assaying for the ability of the column eluate to catalyze hydrolysis of an Arginine-Valine peptide bond in an angiotensin peptide (e.g., AIII), 35 and subsequently conrllllling that the seq~l~nce of the product of the reaction has a Valine residue at N-tenninal amino acid and an AIV peptide sequence.

) 94/00492 2 1 3 9 1 0 5 Pcr/us93/o6o38 The novel AIV ligand-AT4 receptor system of the invention is useful in a comple,..~ ,y or antagonistic role to AII in metli~ting long-term effects of - angiotensins, and in mod~ ting the effects of AI, AII, or AIII on cells. Although not being limited by any particular theory of action, it is believed that: 1) AIV is derived 5 from AII (or AIII) directly (e.g., through the action of a specific AIV angiot~n~in~ e7 and other peptidases); 2) AIV is very labile and will accnmlll~te at physiologically ~ignific~nt concentrations only when high levels of AII are present at the target site;
3) the AT4 receptor is specific for AIV ligand (with accGll,,ually;ilg low affinity for the parent peptide, AIII). Under certain conditions, AIV begins to acc lm--l~te at 10 angiotensin target tissues as the AII levels rise. When AIV concentrations rise to near 0.5nM (i.e., the Kd Of the receptor) auxiliary processes which modify the acute action of AII will be ~ng~ge(l These actions will be mç~i~ted by an intracçll..l~r ~ign~ling system(s) di~lelll from that employed by AII. The activation of such an intracelll-l~r system may potentiate or antagonize the target cell's short-term response to AII. One physiological function of the AT4 receptor-ligand system may be to impart a longer-term response to high-level or chronic angiotensin stim--l~tion in a tissue. Studies support the hypothesis that the AIV ligand-receptor system possesses the characteristics set forth above and is, thel~fole, in a position to serve a short- or long-term modulatory role on the activities of AII. Studies using bovine adrenal tissues have shown that the AT4 receptor is specific, with almost no affinity for AII. In addition, AIV is metabolized/hydrolyzed in bovine adrenal homogen~tes at 200 times the rate of AII and 4 times the rate of AIII. Data suggest that AIV may be derived directly from AII by action of a dipeptidylaminopeptidase, termed herein AIV
angioten~ln~ce.
The location of AT4 receptor sites in groups of cells in tissues allows a skilled artesian to predict likely functions for the AT4 receptor in dirrelc;l" tissues. In addition, it will be recognized that many activities previously attributed to the action of AII (and/or AIII) may be triggered or re~ ted instead by the AT4 receptor-ligand interaction. For example, it is likely that AIV ligand acts as a negative-feedb~c~ agent thus enabling tighter control on the aldosterone release process. The AIV ligand-receptor system may also be associated with a previously inexplicable up-regulation of the angiotensin receptor seen following chronic AII exposure of cells in vi~ro. Still other functions attributed to AII that may be medi~ted instead by the AIV ligand-receptor system include altering the release of catecholamines from adrenal medllll~ry cells or re~ ting adrenal blood flow. It is, the,erore, likely the AIV ligand-receptor system modulates (i.e., increases or decreases) either the acute and/or the long-term WO 94/00492 PCr/US93/0603 2~39~S -24-synthesis and release of chromaffin catecholamines, e.g., by acting to stim..l~te intracell~ r ~Apfes~ion of tyrosine hydroxylase (the rate limiting enzyme in thesynthetic pa~ ar) E~e~ ,enls described below de",ons~ e that the AT4 receptor-ligand 5 system may have a role as a metli~tor of long-term angiotensin effects on endothelial cells (e.g., cell growth; Example 5). AIV ligand-receptor interactions also appear to activate processes in endothelial cells that are comple",e"l~y or antagonistic to those activated by AII. For ;,.sl~llce, some of the AIV ligands that are embotlim~nts of the invention are useful for increasing blood flow (e.g., renal blood flow as demonstrated 10 in the e~"~les).
Because of the widespread distribution of AT4 receptors in many tissues (see examples, below) it is impossible within the scope of this specification to detail every one of AIV's actions in angiotensin-sensitive tissues. However, l~presenla~ e data is provided in the following examples (below), for the physical characteristics of AT4 receptors (see esp. Example 1), for AT4 receptor tissue distribution and speciesdistribution (see esp. Example 2), for physiological functions of AIV peptide ligands and AT4 receptors in controlling renal blood flow, for the cellular biology of AIV
ligand-AT4 receptor interactions (e.g., second messPnger pathways, G-proteins, phosphorylation, intrac~ll--l~r Ca++, phosphoinositide turnover, and guanylate cyclase activity), for vascular effects on venular and aortic endothelial cells and vascular smooth muscle cells and G-protein linkage of certain AIV-receptors, for endocrine effects on adrenocortical cell catacholamine release for effects on cardiac myocytes (i.e., cardiocytes), and for characterization of brain AT4 receptors (e.g., in hippocampal cells and in cerebellum, hippocampus, pi,irO",. cortex, Par 1/2, Fr 1/2, caudate put~m~n, HDB, th~l~mlls, and inferior cnll--c--l-ls), as well as, neurological effects of intracelel~ rentricular injection of AIV (e.g., on le~l~ing and memory).
The disclosures made herein for assays and processes relating to endothelial cells, adrenal cortical cells, cardiac myocytes (cardiocytes), and vascular smooth muscle cells are ~i~cussed briefly below.
As sho~,vn in the examples AIV is active in endothelial cells in ~nh~n~in~
cellular proliferation (as evidçnced by thymidine incorporation) and stim--l~ting production of endothelial cell relaxing factor (EDRF). These results also show the non-interaction of G-proteins with vascular AT4 lece~,lo.~ in bovine aortic or colon~y venous endothelial cells. The results set forth in the Examples further identify a role for the AIV ligand-AT4 receptor interactions in triggering normal and/or hyperplastic growth of endothelial cells in sites of tumors or traumatic or ) 94/00492 21 391 OS PCI/US93/06038 wound injury, and angiogenesis, and a therapeutic use for AIV analogs, agonists,antagonists, and derivatives and covalently modified AIV peptide ligands that are capable of inhibiting vascular smooth muscle cell growth in such hyperplastic states while at the same time promoting endothelial cell growth. The agonist compositions 5 are also useful for encouraging endothelial cell growth, e.g., in wound sites;~nt~goni~t~ for discouraging vascularization in tumor sites. In addition, the AT4 receptor-ligand system may play a role in triggering vasodilation through a selective effect on subpopulations of endothelial cells that exist in particular vascular beds (e.g., in the heart, lung, liver, kidney, brain and the like). As shown in the examples, 10 increased renal blood flow occurs in rats following infusion of AIV ligands and taken together with the demonstrated ability of AIV to stim~ te EDRF production in vascular endothelial cells, the AIV ligand-receptor system metli~tes actions of angiotensin that fall within the bounds of cardiovascular regulation and body water homeostasis. Thus, Ill~;l~c;~ltic uses for AIV analogs, AIV agonists and antagonists, 15 and derivatives and covalently modified AIV peptide ligands include promoting renal blood flow (e.g., in chronic kidney (liqe~ces) or, alternatively, inhibiting renal blood flow (i.e., using inhibitors and ~nt~goni~ts of AIV), e.g., in conditions of hyperacute renal dysfunction and water loss, or during renal surgical procedures.
In cardiac myocytes (also termed herein "cardiocytes") it has been specnl~ted 20 previously that angiotensin II may somehow be involved in the development of left ventricular hy~elllophy since patients treated with angiotensin converting enzyme (ACE) blockers to block blood pressure ~h~l~gec show less tendency to develop left ventricular hy~elLlophy (2~,26). As shown herein, AIV antagonizes the hy~ ophic action of AII. Accoldhlgly, the control of cardiocyte growth may be re~ ted 25 endogenously by a balance between the activating action of AII and the inhibiting action of AIV. It is further believed that AIV and AIV agonists will be effective in blocking the development of, and reversing the effects of, left ventricular hylJel Llophy in p~ti~nt~ Additionally, it is believed that the action of ACE inhibitor is due not to - their inhibition of AII synthesis but to their ability to enh~nce the synthesis of AIV
ligands such as results from the cl.. ~ of p.e~;ul ~u- ~ from the AII synthetic PalLW~r into the AIV palllway. Contrary to current popular belief, the b~-nefi~i~l effect of ACE inhibitors in lle;&~ g cardiac hypertrophy may be due to ACE inhibitor ~nh~nc~m~nt of the formation of AIV.
The data presented herein also indicates that AII and AIV operate by separate 35 receptors employing di~erellL intr~cçll-.l~r ~i n~ling systems. It has been reported that ACE inhibitors may have a beneficial effect in red~ing cardiac hypertrophy through WO 94/00492 2J~39~S PCI/US93/060 effects at the level of AII or AIII. Considering the results disclosed herein it is most likely that the long-term effects previously attributed to decreased AII may in fact be m~di~ted by the interaction of increased levels of endogenous AIV ligands with the AT4 receptor. Further, it is most likely that the antagonists and agonists of AIV
5 ligands, disclosed herein, will provide improved pharm~ceutical compositions for treating cardiac hypelLlophy attributable to the renin-angiotensin system, e.g.
ventricular hyl~clllophy. The inventors believe that the interaction between AIV and the AT4 receptor may trigger the receptor and inhibit growth in cardiomyocytes.
In adrenal cells angiotensin AII's role in the regulation of aldosterone release10 from the adrenal cortex is reportedly well established (27). As shown herein, certain activities (such as adrenocortical cell growth), previously attributed to AII or ATI, are actually activated following AIV ligand binding to the AT4 receptor. AII (and AIII) reportedly stim..l~tes aldosterone release from adrenal glomerulosa cells. The disclosure, herein, of high levels of AT4 receptors in adrenal cortical cells 15 (E~ples 1-2) sllggests a possible role of AIV ligand (i.e., rather than AII or AIII) in triggering AT4 receptors on adrenal cells to inhibit AII-m~di~ted aldosterone release.
Another role of AT4 receptors in adrenocortical cells may be to up-regulate the threshold level of AII ligand required to trigger a cellular response by re~ ting the levels of cellular AT1 and/or AT2 ccel~o~ and/or to regulate adrenal blood flow.
In addition to being found in high concentrations in the adrenal cortex, AT4 receptors are found at even higher levels in the adrenal n1e~Ullnry cells where AII has previously been reported by others to potentiate catecholamine release. AIV ligand may modulate release of catecholamines (i.e., increase or decrease the release) acutely (or possibly even long-term, e.g., by triggering the AT4 receptor and thereby stiml~l~ting increased or decreased t~-t;ssion of tyrosine hydlo~ylase, the rate-limiting enzyme in catecholamine sylllhes;s.
In vascular smooth muscle cells the role of AIV and its specific receptor appears to be similar to that articul~ted above for AIV in cardiocytes: AIV may act to inhibit growth of the cells thus opposing the action of AII. Agonists of AIV binding to the AT4 receptor will be effective inhibitors of vascular smooth muscle growth and will be ~Lc~ ;c~lly useful in reducing neoillLilllal growth which often occurs following angioplasty.
As disclosed herein, high levels of AT4 receplol~ are present in cardiac and vascular tissue, inclurling cultured bovine endothelial cells. The disclosure, herein, that AIV ligands and the AT4 receptors may function as growth factors of the tyrosine kinase class indicates that certain inhibitors of tyrosine kinase growth factors 'O 94/00492 . j ' PCr/US93/06038 -27- .

may also serve as inhibitors of certain angiotensin AIV ligand-receptor system-medi~ted cellular hy~tlLlophiC processes (e.g., ventricular hy~cl~lophy), and that nucleotide probes constructed for compl~ y to portions of RNA encoding the AIV ligand and receptor sequence may be useful in idcllLiryiilg other members of the - 5 AIV family of growth factors.
The invention also provides diagnostic applications for the AIV peptide ligands and antibodies. The role of the AT4 receptor-ligand system in cardiovascular regulation suggests a possible value to diagnostic tests for monitoring the levels of AIV ligand and AT4 receptor in biological fluids and tissues (i.e., rather than AII or AIII). Individuals with high renin-sodium profiles are reportedly at five times greater risk of myocardial infarction than individuals with low renin-sodium profiles despite adequate control of systemic blood pressure (28).
The AIV peptides, ligands, receptor fr~pmP.ntc, and the like disclosed herein are useful in diagnostic assays, e.g., immllno~cs~ys~ for the detection of the presence or ~molmtc of AIV ligands or receptors in tissues, cells, and biological fluids of patients. The AIV peptides, ligands, analogs, derivatives, or covalently modified AIV
peptides of the invention may be form--l~ted in buffers with stabilizers, e.g., for use as positive or negative controls in diagnostic assay, or in reagent test kits for receptor-binding assays.
Those skilled in the art will recognize that the AIV ligands of the invention may be readily employed using conventional techniques to produce polyclonal or monoclonal AIV ligand specific antibodies, and that the isolation and purification of the AT4 receptor provides materials useful for prepal~ion of polypeptide fra~n~.ntc (e.g., using CNBr and proteolytic enzymes) that can be subjected to automated amino acid seq~lenring The amino acid sequçnce of the AT4 receptor, in turn, provides the sequence data neces~,~.y for construction of conserved and degencl~le nucleotideprobes for cDNA or genomic moleclll~r cloning of nucleic acids e,.~lessing the AT4 receptor, mutant AT4 receptor, or fragm~ntc of the AT4 receptor. A convenient method for molecular cloning of the receptor is provided in Example 7.

Physical Characterization of the AIV Receptor Kinetic bindin~ studies: bovine adrenal cortical Illclllbl~es In kinetic binding studies, con-lucted as set forth in Example 1 Materials and Me~hods described below, both l25I Sarl,Ile8-AII and 125I-AIV binding were characterized by slow association rates (kl=1.01+12x10-2 and 5.58+0.64x10-2 nM~lmin~l, le~,e~ ely), very slow dissociation rates (k l=2.36~0.49x10-2 and WO 94/00492 ~39~5 PCr/US93/0603~

2.57_0.05x10-2 nM~l min~l, respectively), and high affinity binding (calculated Kd=2.25+0.26xlO-1M and 4.42_0.46xlO-1M, respectively; number of w~ LS
(n) = 4) (Table 2).

S Kinetic consL~ s for l25I Sarl,Ile8-Ang II and l25I-AIV binding to bovine ~drenal cortical mtlllbl l1es. *
T ig~nrls kl (nM~l min~l) k l (min~l) K~(M) 5I Sar1,Ile8-Ang II 1.01 + 12x10-2 2.36 + .49x10-2 2.25 + .26xlO-10 l25I-AIV 5.58 + .64x10-2 2.57 + .OSx10-2 4.42 + .46xlO-10 * n = 3, mean + SD
Equilibrium binding studies: bovine adrenal cortical lllelll~ es Equilibrium binding studies were contl~lcte~ to evaluate the binding of l25I-AIV to receptors in bovine cortical membrane plep~lions. Comparisons were made of the binding of both AIV and of AII, i.e., to the c~ ic~l ATl receptor sites defined by binding of l25I-Sarl,Ile8-AII. Binding studies were carried out in buffer con~ -g SmM EDTA, lO~lM Bestatin, SO~M Plummer's inhibitor, and lOO~M
PMSF, developed specifically to inhibit metabolism of angiotensin fr~gmPnt~ and l S receptors during the assay.
Saturation isotherms for 125I-AII and 125I-AIV indicated the presence of two distinct separable high-affinity binding sites in bovine adrenal cortical me",bl~ne plep~ions, i.e., one for AII ligand and a second for AIV ligand. The equilibriumconsL~s calculated from this data were as follows: a) for AII receptor-ligand binding (i.e., 125I-Sar, lIle8-AII) the Kd=0.54_0.14nM, Bmax-1.03_0.26pmol/mg membrane protein (n=4); and b) for the. AT4 receptor ligand binding (i.e., l25I-AIV) the Kd=0.74_0.14nM, Bll,ax=3.82_1.12pmol/mg membrane protein (n=4). The results of the equilibrium binding studies with ,llelllblai1e-bound AT4 receptor are nn~i edinTable3.

V 94/00492 j ~ 21 39 1 05 PCr/US93/06038 Equilibrium Binding ConsL;a,~ls for 125I Sarl, Ile8-Ang and 125I-AIV Binding to Bovine Adrenal Cortical Mel.,bl~es.
sma~ Hill Ligand K~l (nM)(pmol/mg prot ) Coeff. r(Scatchard) 125I Sarl, ILe~-Ang II 0.54 _ .141.03 + .26 1.00 + .03 0.91 + .08 125I - AIV 0.74 + .143.82 + 1.12 1.00 + .02 0.93 + .05 n=4, mean + SD
The results of these kinetic and equilibrium binding studies show: (a) two separable high affinity binding sites, one for AII and a second for AIV; (b) large di~erences in the ...s.x;...~l binding (BII~ax) per mg ...~-..b-~ e protein, i.e., with more than three-fold more AT4 receptor in this p~ep~a~ion than AII receptor; and, (c) no cross-~ pl~c~m~nt of AII binding by AIV or vice versa. The results provide 10 convincing evidence for the existence of two separable receptors; one for AII and a second for AIV. However, the theoretical possibility existed that a single receptor might have di~-ing affinities for AII and AIV. Since it was known that ATl and AT2 are commonly destroyed during extraction from .llelll~ es, and are also heatlabile (i.e., at 60C) a scheme was devised to rule out the latter possibility by testing 15 for AT4 leceplo-~ in solubilized and heat-treated men-b.~le plep~alions The results of these studies are presented below.
Equilibrium bindin~ studies: solubilized bovine adrenal cortical AT4 receptor Initial studies, con-lucted as described in Example 1 Materials and Methods, col~fi....ed that 125I-AIV bound to solubilized receptors in ,-.e...b-~1e p.~p~lions 20 which would not be expected to contain ATl or AT2 receptors. The kinetics of binding of l25I-AIV to the solubilized bovine adrenocortical receptor, at an AIVligand conce.~ lion equal to 25% of the appa,~,.ll Kd (with 25~g of .llcll.b.~e protein), in~lic~ted that equilibrium was reached in appro~i-..alely 100 min. at 37C.) The plateau region of binding to the solubilized receplor for 125I-AII or 125I-AIV, (after reaching equilibrium), was stable for at least one additional hour. The off-rate of the AT4 receptor, as de~ . i-.ed following the addition of 1000-fold excess of unlabeled AIV, was c-ceedil-gl~ slow, with an average tl/2 =292.4 min (n=5).
Equilibrium binding studies were next cond~cted at 37C with a 120-minute incubation (as in the Materials and Methods) with the solubilized .llell~b-~l1e receptor preparations. Saturation isotherms for 125I-AIV (Figure 2A) and 125I-AII (not shown) were developed to cGlllpare the equilibrium binding constants of the WO 94/00492 ~i r~ 213D 1~ ~ 30 PCI/US93/0603~

solubilized AT4 receptor. A concentration range of about 5 x 10~M to about 5 x 10-12M AIV was employed in a typical cA~Jelilllen~ using 25~g of total protein.
The best fit for the l-ansrulll,ed data using the LIGAND program revealed a single AIV binding site with no apparel-L cooperactivity. A sumrnary of the binding data for 5 AIV ligand to solubilized receptor is found in Table 4.

Equilibrium Binding Data for 125I-AIV to Bovine Adrenal Cortical Solubilized Receptor.
*K~(~ Bm~"~(fmol/mg protein) r(Scatchard Plot) Hill Coefficient 5.06 ~ .57x10-1 87.9~9.7 0.991 ~.009 0.995~.039 *N=4, mean ~ SD
The data presented in Table 4 shows that the solubilized receptor, like the e.llbl~e receptûr (Table 3), has an extraordinarily high binding affinity for AIV.
Collll)eliLion binding studies: bovine adrenal cortical melll~ es To establish the specificity of the AT4 receptor, colll~,e~i~ion curves were developed with several diLrelell~ angiotensin analogs using a concentration range of 15 10~M to lO-llM. Co...p~i~ons were also made of the binding specificity of ~l~c.ci~
AT1 rèceplor binding sites (i.e., 125I-Sarl,Ile8-AII binding sites). Competitionanalysis (the su.. ~i~ed results of which are presented below in Table 5) also clearly ~lictin~liched the eyict~onre of two distinct rèceplol~ based on their specificity for di~re..L ligand structures in the angiotensin analogs. (The r values for log-logits tran~r~,-l--alions of the competition data were typically ~0.98.) Binding of 125I-AII
ligand to the AII receptor (as characterized by binding of 125I-Sarl,Ile8-AII) was effectively cGllll)tlili~rely h~hil,i~ed by Sarl,Ile8-AII, AIII, and DuP 743. In contrast, AIV ligand, AII(4 8~, and CGP42112A delllol1sll~led very little affinity for the AII
binding site (Table 5.) The pattern of binding at the AII site is consistent with a Type I classic AII binding site (20,25). (Binding Sarl, Ile8-Ang II, Sarl, Ile8-Ang II, AII, AIII, and DuP 753 with high affinity is a pattern of binding specificity consistent with an ATl site.) In contrast to the AII lece~Jtor~ the binding site for 125I-AIV
ligand was effectively cGl~elili~rely inhibited only by AIV ligand and to a lesser extent by the peptides in the AIII prepa~lion (Table 5).

'~0 94/00492 ~ ~ -31~- 1 0 ~; PCI /US93/06038 Competition of l25I-Sarl,Ile8-AII and 125I-AIV Binding to Bovin~ Adrenal Cortical Men~ nes.
l25I-Sarl,IIe8-AII 125I - AIV
Competitor Binding (Kj, M) Binding (Kj, M) Sarl,Ile~-AII 0.22 + 0.10 x .10-9 >10 AII 2.01 + 0.67 x .10-9 >10~
AIII 1.15 + 0.34 x .10-9 >14.50 + 2.3 x 10-9 AIV >10~ 0.58 + 0.15 x .10-9 AII(4-8) >10~ >10~
DuP743 3.10 + 0.67 x 10-8 >10-4 CGP 42112A >10 1 >10 1 Binding studies: two receptor binding states in rabbit heart melllblanes S Studies were next conducted to f-;.. ;.. e the kinetic p~n~,Lers of125I-angiotensin IV binding to receptors in P2 mwll~ f prep~lions from rabbit heart. Col~ ;sons were made of the binding of both AIV and of AII, i.e., to the classical AT1 recf~or sites defined by binding of 125I-Sarl,Ile8-AII. Binding studies were carried out in a buffer (below) co~ ",ng an extensive cocktail of inhibitors that 10 was designf,d to minimi7e metabolism of both the receptor and the test ligand, i.e., the buffer co~ ned 5mM EDTA, 0.2% BSA, lOIlM Bestatin, 5011M Plummer's inhibitor, and lOO~MPMSF.
Angiotensin peptides (i.e., AI, AII, AIII, or AIV) were stable in this buffer for

4 h at 37C with less than 10% hydrolysis measured by reverse phase HPLC.
The studies were con~lcted as described in the Materials and Methods, below. The association rate COIlsl~lt (kl) for 125I-AIV was dele"",i1ed to be 3.05 x 108M-l min~1 and the dissociation rate consL~,I (k l) was 0.028 +/ 0.017 min~l The overall dissociation con~ (Kd) measured under equilibrium binding conditionswas determined to be 9.15 x 10-11M. (The results rep,~st;llt the mean values from the 20 results of 4 expe"",~ con~ucted using clllplicate s~mrles ) Saturation isotherms and Scatchard analysis produced data best resolved in a two-site model using non-linear curve fitting n ethod~ (LIGAND p[og,~., curve fitting options). The Kd for site #1 was determined to be 10.3 +/- 3mM with B~ =1747 +/- 393 fmoVmg; the Kd for site #2 was 10.1 +/- 5pM with BmaX=l 5 +/- 4 fmoVmg. Binding to the rabbit heart 25 mtlllbl~nes was competitively inhibited in a specific manner by AIV but not AII, AIII, 125I Sarl,Ile8-AII, DuP753, CGP, AII(4 8~, or DAAI (see Table 6).

Wo 94/00492 PCr/US93/06038 Coll")t;Li~ on of Binding of l2sI-AIV to Rabbit Heart Membrane Receptors Competitor Kj (M) AIV 1.4 x 10-9 AII >10~
AIII 2-3 x 10-7 Sarl ,Ilef~-AII >10 DUP753 >10 CGP >10 AII(4-~) >10~
DAAI 90.5 x 10-9 The data in Table 6 was s~lc~ ted from colllp~Lilion displ~cem~.nt curves for binding of 0.5nM 125I-AIV to ,l,elll~l~l e fractions; lll~;lllbl~u~es were inr,ub~ted for 120 min. at

5 37C in the presellce of lOpM to lmM co,llpeli~or; possible conversion of AIV to other (smaller) fr~nrntc was evaluated after 120 min at 37C by adding 20% TCA to stop the inc~lb~tion, and then ev~ ting the pelcell~age of AIV by reverse-phase HPLC on a C18 column with a 20% ACN/TEAP3 mobile phase; greater than 92% of the 125I label present at the conclusion of the inc~lb~tion was present as AIV.
10 Structural re4uiltlll~ s for ligand binding to bovine adrenal AT4 receptol~
The results in Table 7, also include a Sullllll~y of studies ~Içcigned to analyze the structural realules of the N-terminus of an AIV ligand that are required forbinding to an AT4 l~ceptor. The results of these structural studies are also presented in Figure 2B. The results show that modification of the N-terminal valine residue 15 (i.e., by N-terminal shortening of AIV to AII(4 8)), or ext~n-ling the N-terminus with a hydrophobic residue such as Sar or GABA, or c.l~ gil-g the stereoisomer of the L-Val to D-Val, all drastically decrease binding of an AIV ligand to the AT4 receptor (Table7). The AT4 receptor also failed to bind DuP 743 (DuP, Figure2B) or CGP 42112A (CGP, Figure 2B) and thus did not exhibit the pharmacological 20 ~rop~,lies of a classic AII binding site (26). As shown in Figure 2B, the ability of the various compounds to inhibit AIV binding to the solubilized AT4 receptor was tested.
The following compounds are shown in Figure 2B as follows: DAAI1, desAsp angiotensin I (i.e., idçntic~l at the N-terrninus to AIII; see open squares with a dot, Figure 2B); AIV, angiotensin IV (closed di~montlc~ Figure 2B); AII (open squares, 25 Figure 2B); SIAII (AII lacking the Ile residue at position 5, Figure 1; open diamonds, Figure 2B); DuP (Dup 743, an AII analog; open squares, Figure 2B); CGP
(CGP42112A, another AII analog; closed triangles, Figure2B); and, AIII (open triangles, Figure2B. The results presented in Figure2B and the Ki values ~vo 94/00492 2 1 3 9 1 0 S PCr/US93/06038 -33 . ~

summarized below in Table 7 show that: (a) only AIV, and peptides in the DAAI1 (i.e., AIII N-terminal sequence), and AIII plep~ions effectively competitively inhibit binding of the 125I-AIV ligand to the AT4 receptor; and (b) the peptides in the AIII, Sarl-AIII, and DAAI1 preparations are appro~hl~ately 100 times less effective than AIV in colllpe~illg binding to the AT4 receptor.

~, ~13~0S

Competition of 125I-AIV Binding to Solubilized Bovine Adrenal Cortical Receptor.
Analog/Competitor Kj (M) AIV 5.67 + 1.71 x 10-1 AIII 2.28 + 0.17 x 10-8 AII > 10 AII(4~ 10 d-Vall AIV > 10 Sarl, Ile~-AII > 10~
Sarl-AII 2.80 + 0.41 x 10-7 Sarl -AIII 8.25 + 0.52 x 10-8 Sar~-AIV 1.44 ~ 0.47 x 10-7 GABA-Nterrn-AIV > 10 des Phex-AII > 10~
DuP 753 > 10-4 CGP42112A ~ 10~
des Phe,~-AIV 7.45 + 0.96 x 10-8 AI(~-ln) 9.63 + 1.02 x 10-1 Me-Y-I amide 2.06 x 10-9 ~ 1.59 x 10-1 Me-Y-I 8.30 x 10-9 + 1.80 x 10-9 Me-Y-I-G 8.75 x 10-9 + 1.67 x 10-9 Heptanoyl-Y-I 1.96 x 10-8 + 2.42 x 10-9 KYI 3.00x 10-8+9.67x 10-9 Me-F-I 5.17 x 10-8 + 5 90 x 10-9 KallVal~ AIV 1.03 x 10-9 + 2.21 x 10-1 VallVal~ AIV1.29 x 10-9 + 9.10 x 10-11 Kall AIV 2.13 x 10-9 + 8.93 x 10-1 Val~ AIV 1.01 x 10-8 + 5.62 x 10-9 Vall AIV 1.69x 10-8+5.09x 10-9 D-Vl AIV 9.68 x 10-7 + 5.59 x 10-8 D-Y,AIV 4.16x10-7+7.69x10-8 D-I~ AIV 5.82 x 10-7 + 2.60 x 10-7 D-H4 AIV 2.28 x 10-9 + 7.92 x 10-1 D-P~ AIV 2.32 x 10-9 + 7.31 x 10-1 D-F,~ AIV 1.18 x 10-9 + 5.62 x 10-10 Gl AIV 1.68x 10-7+2.19x 10-8 G~ AIV 1.00 x 10~ + 8.39 x 10-9 G~ AIV 1.16 x 10-7 + 1.43 x 10-8 G4 AIV 6.98 x 10-1 + 6.10 x 10-G~ AIV 2.10x 10-1+2.03 x 10-G,~ AIV 7.16 x 10-9 + 1.18 x 10-9 *N=4, mean + SD

., .

YVO 94/00492 21 391 o;~ Pcl/US93/06038 The combined results show the importance of the valine at the 3 position for binding of an AIV ligand to the solubilized bovine adrenal gland AT4 receptors (i.e., D-Vall; des-Vall-AIV are inactive). The results also show the apparell~ in~ignificance of Pheg for AIV binding to this AT4 receptor (i.e., desPhe6-AIV still retained AIV
binding activity). Thus, a minim~l AIV penta peptide ligand having a sequence VYIHP retained AT4 receptor binding activity.
For high affinity binding of an AIV ligand to an AT4 receptor, the structure of the N-terminal neutral polar amino acid (e.g., valine) is most important. N-terminal extension is inco~ alible with binding, deletion of the terminal valine residue ç~ es binding (Ki ~10-6), substitution with Sar decreases binding affinity, substitution with Phe results in a 5-lO-fold decrease in the affinity of binding, Pro-substituted AIV peptides bind with 100-fold lower affinity, but substitution with Ile results in equivalent binding, and Lys substitution results in lO-fold higher binding affinity of the KYIHPF AIV ligand (data not shown).
In the studies presented in Tables 6-7, and Figure 2B (abpve), a minim~l level of binding of peptides in AIII prep~ions to the AT4 receptor was observed. This most probably It;plesen~s an artifact in the assay and/or ligand plel)al~ions that is easily accounted for by a low level of N-terminal hydrolysis of AIII to AIV, e.g., the appalellL affinity of peptides in Am plt;lJ~alions for the AT4 receptor (i.e., at 22.8nM) can easily be .oYpl~ined by ap~Jlo~ lely 2.5% hydrolysis of AIII to AIV.This notion was supported by the results of studies cond~lcted with density-gradient-purified heat-treated ll,t;lllbl~-es (i.e., heat-treated and purified to minimi7e en_ymatic activity in the Ill~lllbl~le plepa.~ions). In these studies the amount of AIII
conversion to AIV was substantially reduced and the apparellL affinity of AIII for the AT4 fecel"or was also reduced (Ki=29.3~3.3x109M), i.e., from appr~illla~ely 29-fold less active than AIV ligand (above) to about 52-fold less active (here). This supports that notion that hydrolytic cleavage in the assay (i.e., medi~ted by a specific cell Illt;llll)l~e AIV angiotç~ e or by another non~ecific peptidases) can convert an AI, AII, or AIII peptides to an AIV ligand, and that low levels of AIV ligand in these ple~&la~ions accounts for their appare.l~ binding to the AT4 receptor.
Additional support for this notion is provided by the results of studies (data not shown) which show that the percellLage hydrolysis of AII or AIII in a preparation correlates with the effectiveness of a given ple~ Lion as an inhibitor of AIV ligand binding to the AT4 receptor. In this study pl epal ~Lions of AII or AIII were incubated at 37C for dif~lenL periods of time and (i.e., x% AIV) the extent of hydrolysis to AIV was detellllined by reverse phase HPLC (i.e., x% AIV). In every in~t~nce 100%

O 94/00492 PCr/US93/0603 of the apparent binding of l25I-AII or l25I-AIII was due to actual binding of 125I AIV.
As further shown in Table 7, the D-substitution and glycine-substitution data confirms that positions 1-3 of the AIV molecule are critical for determining binding 5 affinity to the receptor. Positions 4-6 are less critical. In fact removal of C-terminal groups appears to ~nh~nce binding affinity perhaps by redu-~.ing steric constraints.
T.ig~nds co~ g C-N nonpeptide bonds can be produced that possess high afflnity.
In general, highest affinity is obtainable by dual modifications at bonds between amino acids 1-2 and 3-4. Val(l)Val(3) AIV appears totally resistant to en_ymatic 10 degradation upon exposure to rat kidney homogenates. As further shown in Table 7, tripeptides CG~ g straight chain aliphatic amino acids in position 1 exhibit high affinity. To date, the highest affinity is achievable with the Me-Y-I amide, suggesting that amides are preferable to free acids and that the chain length found in Nle is optimal (both longer and shorter bind with lower affinity).
Note that a similar approach to that taken with the N-terminus is useful to characterize the C-terminal. Stereoisomers (e.g., D-Phe), C-terminal extension, and sequential C-terminal deletion, and other peptide analogs can be synth~ei7ed andtested for their ability to co...~,eli~ ely inhibit with 1251-AIV binding to purified bovine adrenal melllbl~les. If C-terminal extended peptides are found to bind to the 20 receptor, appropliate C-terminal extended peptides can be constructed for use in the affinity chlc,llla~ography purification of the receptor. Substitutions and modifications of internal amino acids can also be ~Y~mined for their effects on binding. Once physiological or second messçnger systems have been id~ntified (Example 6) as being AIV-dependent events, then they can be used for drug screelf~ng, and a second round 25 of synthesis can co. "~ nce focusing on the development of receptor antagonists.
The materials and methods and eA~tlh~,tn~al assay conditions employed in Example 1 are described below:
Materials and Methods:
Peptide Syll~hesis The angiotensin analogs employed in this study were synth~.ci7ed by the sl~dald Merrifield method utili7ing t-Boc protected amino acids and chlolc,lllt;lllylated resins on a Vega250 coupler a~lolllaled synthPci7er Following synthesis, the crude peptides were purified by prep~a~ e reverse-phase HPLC using a 1 h gradient for elution at 9ml/min. Initial conditions were 90% H20, 10%
ace~oni~lile, and 0.1% TFA and the final conditions at the top of the gradient were 65% H2O, 35% acetonitrile and 1% TFA. Purified peptides were amino acid .

~O 94/00492 2 1 3 9 1 0 ~ PCr/US93/06038 analyzed to determine both peptide and total purity. Typically the peptides produced were greater than 99% pure and contain 20-25% acetate.
Tissue Plèpal~lion: bovine adrenal cortical tissues Adrenal cortex was removed from bovine adrenals obtained from a local 5 s~ ghtçrhouse. The minced cortex was then homogenized in a Polytron as a 40:1 suspension in assay buffer at 10 sec/ml. The homogenate was then centrifuged at 500g for 10 min. to remove whole cells and nuclei. After a rehomogenization and rece~lLlir~lgation the combined supellla~ s were spun at 40,000 x g for 20 min. The pellet was rehomogenized and respun at 40,000 rpm for 30 min. This final pellet was 10 resuspended in assay buffer and layered on a discontinuous sucrose gradient (0.8M/1.2M). After a 100,000 x g spin for 90 min. the purified membranes were located at the density interface and were removed. The sucrose COI~ g Illelllbl~le suspension was diluted 1:10 in assay buffer and spun a last time at 40,000xg for30min. The pellet was resuspended in assay buffer at a concenLl~Lion of 10mg protein/ml and heat treated at 60C for 30 min. in the plesellce of 20mM MgCl2. The llle~llbl~1es~ now devoid of almost all peptidase activity, were ready for use in the binding assay.
Bindin~ studies: bovine adrenal cortical melllbl~1es To test the ability of the synthesi7ed analogs to colllpeLiLively inhibit for 20 l25I-AIV binding a tii~pl~r,Pmrnt curve was established using purified bovine adrenal cortical lllelllbl~es. Binding was carried out in 10-75mm siliconized glass culture tubes CG..~ g 0.2nM l25I-AIV, 25mg of membrane protein, and the desired analog over a concentration range of 10-l2M to 10 4M using half-log dilutions. All binding inr,ub~tions were carried out in duplicate at 37C for 2 h in a buffer co-~ g:
50mM Tris, 150mM NaCl, 5mM EDTA, 10~M bestatin, 50~M Plummer~s ~e~grnt, lOOIlM PMSF and 0.2% BSA (assay buffer) in a total volume of 0.25ml. After incub~tion, the inr,ubates were filtered through GF-B filters soaked in 0.3%
polyethyle.~e;...;..e and washed with four~ml washes of PBS. The filters were then counted in a BeçL-m~n 5500 gamma counter. A typical e~ elilllenL rX~mines 5 analogs simlllt~neously and inr,ludes a positive control curve in which AIV was used as the displacer. All curves were run in quadruplicate, each with a dirrèlellL tissue pre~ aLion Nonspecirlc binding was defined as total binding minus binding observed in the presence of 100mM Sarl,Ile8-AII or 100mM AIV. No cross displ~crmrnt (i.e., of AII binding by AIV or AIV by AII) was observed. HPLC
analysis of both the bound and free 125I-AII or 125I-AIV ligand intlic~ted that 100%

W O 94/00492 PC~r/US93/0603 ofthe "specifically bound" label was either l25I-AII or 125I-AIV, respectively, and the overall hydrolysis of l25I-AII under conditions of the assay was less than 2%.
Data were analyzed by the LIGAND program (29) from which Ki values can be obtained.
5 Binding studies: solubilized bovine adrenal cortical receptor Solubilization and characterization of the receptor from bovine adrenal Illelllbl~nes was accomplished by homogenizing the ~ lllblanes (above) in hypertonic buffer followed by fractionation of the melll~ es by sucrose density gradient centrifugation. The Illelll~l~e pl~p~Lion was then heat treated at 60C in the 10 presence of MgC12 (to inactivate AT1 receptors). The heat LleAtlll~ t also reduced endogenous peptidase activity in the plepal~ions by 90-95%. The AT4 receptor in the prep~lions was then solubilized using 1% zwitterionic detergent 3-[(3-cholamidopropyl) dimethyl ~Ill~lolLo]-l-plu~.Anes~lfonic acid (CHAPS).
Binding studies: rabbit heart P2 nlc;lllbl~1es from rabbit heart were plepared by homoge~ ;on and di~erellLial centrifugation at 4C. Binding was carried out in the presence of 5mM
(EDTA), 0.2% heat-treated bovine serum albumin (HTBSA), lO,uM Bestatin, 50~1M
Plummer's inhibitor, 100~M phell~ ,eLll~lsulfonylfluoride (PMSF), and 50mM Tris,pH7.4, at 22C. Binding was initiAted by the addition of lOOmg protein and 20 applupliale amounts of labeled ligand. (For kinetic binding studies the samples were inc~bAted for 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, and 220 mimltes at 37C. For equilibrium binding studies the same conditions were used and samples were inr,~lbated for 120 min at 37C.) All ;.~ ul~al;ons were c-)ndllcted at a final volume of 250ml in 12 x 75mm siliconized (SigmaCote) borosilicate tubes, and they were 25 termin~ted by rapid vacuum filtration in a Brandel cell harvester through glass fiber filters (schlpichp~r and Schuell #32) soaked in 0.3% polyethyl~-~;...;..~. Filters were immP.di~tely rinsed with 4x4ml 150mM NaCI, 50mM Na2HPO4, pH7.2 at room telll?elaLl~re~ Filters were allûwed to air dry, placed in fresh tubes and counted in a gamma counter. Specific binding was defined as the di~rei1ce bt;Lween the absence 30 and presence of l.OmM ~i~pl~in~ ligand.
Dissociation (i.e., of ligand from lecepLol~) e ~ illlellLs were con-l~lcted by adding lmM unlabeled AIV ligand colllp~tiLor to the assay at 120 mimltes after iniSi~ting binding (at 37C) with 0.5nM 125I-AIV.
Saturation isotherms for binding were conducted with approx;..,~tely 25~1g of tissue protein inr,~lbated with various concentrations of l25I-AIV for 120 min. at ~094/00492 21 391 OS Pcr/US93/06038 -3~-37C; nonspecific binding was defined in the presence of lmM AIV. Three cA~uelilllents were contlllcted resulting in 36 data points for Scatchard analysis.

Tissue and Species Distribution of the AIV Receptor 5 Species Distribution:
A second major approach to dçfining separate and distinct binding sites was to e~mine their relative tissue and species distribution (Table 8). The results presented in Table8 show the fentamoles of AII or AIV bound per milligram of lll~lllbl~e protein in extracts plepared under identical conditions from the tissues and species 10 indicated.

*Dist-ibution of l25I-SI-AII and 12sI-AIV Binding in l~mm~ n Tissues.**
I2sI-AIV
Species Tissue125I sI AII(frnoVmgprot ) (fmol/mg/prot.) cow adrenal medll l"218.8 + 56.2 397.3 _ 53.6 pig whole adrena ND 70.8 ~ 6.7 horse whole adrena 1.8 + 1.0 72.7 + 13.5 dog whole adrenal 4.6 ~ .7 36.5 ~ 5.4 cat who:e adrenal 3.3 ~ 2.3 199.6 + 19.7 rabbit wnoeadrenal 79.6+21.6 105.3 1 15.6 rat who e adrenal158.2 ~ 21.6 N.Det.
guinea pig wnole adrenal 45.6 + 9.2 101.2 + 26.3 cow coronary venule2.9 + 0.3 85.1 + 3.3 endothelial cells rabbit heart 10.6 1 3.6 249.9+36.3 *2511g of .. ~ protein was I with 500,000 cpm of label. Specific binding was defined as total binding no~ ;r.r binding at lOO nM ~ ed peptide.
~"*n = 2~; mean ' SE; l25I-SI-AII=l25I-Sarl,Ile8-AII; fmol/mg ~r ~ ~I~~ (i.e. lo-l5M) of AII ligand bound per mg of total protein in the pr~p ~r:nion N.Det. = not d- - ' 'e, i.e., less than 1.8 fmol/mg protein.
The results show that: a) the human AIV ligand binds AT4 receptors in a wide variety of ".A."".Ali~n species; and b) most --~------~li~n adrenal tissue express an 20 AT4 receptor capable of binding the AIV ~peptide VYI~F.
Tissue Distribution:
In order to begin to assign physiological functions to the AIV ligand-AT4 receptor interaction, plelil.~in&,y tissue distribution studies have been con~lcted in guinea pigs. Guinea pigs were chosen because their adrenal tissues demonstrated high 25 levels of AIV binding (Table 8). The tissue distribution of AT4 receptors was WO94/00492 'L39~0~ PCr/US93/0603~

measured by assaying radioligand binding to ".c"~,~ne plep~ions, i.e., as described in Example 1, above.

Distribution of 125I Sarl, Ile8-AII and 125I-AIV Binding sites 5in the Me",b,~l1es of Guin~a Pig Tissues.*
125I Sarl, Ile8AII 125I-AIV
Tissue Binding (fmol/mg prot) Binding (fmol/mg prot) aorta3.17 ~ 2.2 45.4 ~ 11.0 brain17.70 + 9.5 60.8 ~ 13.5 heart5.70 + 0.6 83.3 ~ 20.8 cidney 8.10 + 1.19 22.7 ~ 12.1 iver22.40 ~ 5.3 28.9 ~ 6.4 lung12.20 ~ 6.4 56.1 ~ 10.1 uterus 4.00 ~ 1.5 87.0 ~ 6.3 *n = 4; Binding was carried out as described in Table 6, above.
The co",bhled results presented in Table 9, above, show that the receptor was widely distributed in the tissues. The co",bi,led results suggest evolutionary conservation of both the AT4 receptor and the AIV ligand.
10Additional studies were next cond~lcted to physically co",p~c the receptors inguinea pig brain, rabbit heart, and bovine colon~-y venule endothelial cells with respect to their binding affinities for AIV ligand. The results of these studies in-lic~te that cells in these di~lcllL tissues all have AT4 receptors with cGlllp~able binding affinity for AIV ligand; each cell type has an AT4 receptor with a Kd for 125I-AIV of 15 about O.lnM to about 0.5nM. Next, cG",~eLilion studies similar to those presented above in Example 1 were con-lucted to evaluate the structural requile",ellL~ forbuilding AIV in di~relll tissues. The results inrlic~te that each AT4 receptor exhibits the same N-terminal specificity recorded, above, i.e., for V, I, or K. The results of these co",~ined studies support the notion that a rclll~kable degree of evolutionary 20 conservation has been ~ ;..ed for AIV ligand-AT4 rec~Lor system, and this level of conservation is cGIll.llonly predictive of i"~pO"~I~, and probably critical, physiological functions.
Surprisingly, it has not been possible to demonstrate binding of 125I-AIV in rattissues incl~din~ brain, heart, kidney, aorta, lung, liver, or cultured smooth muscle 25 cells (n=5) (Table 8). At this time, the ~i~ific~nce of this finding is uncertain. It is possible that the in vitro assay conditions may not be optimal for this species and that the AT4 receptor or AIV ligand may be rapidly inactivated, e.g., by high levels of peptidases known to be present in rat tissues.

`YO 94/00492 21 ~gl OS PCr/US93/06038 In addition, on a technical note, binding of the AIV ligand to the AT4 receptor was inhibited in the presence of Bacitracin (i.e., 10mg/ml; at a final calculated concentration in the assay of 0.07M). Bacitracin is a polypeptide antibiotic with the sequence ICLEIKOIFHDD (i.e., O is ol.lill~ille), and it is often inr,l~lded in angiotensin 5 binding assays to inhibit the action of nolls~ecific proteases (i.e., as an altemative substrate for the proteases). The observation of bacitracin il~le~rt;~ t;nce is of potential significance for at least two reasons: 1) previous investigators who have inrhlded bacitracin in their assay buffers may have inhibited AIV ligand binding to the AT4 receptor; and, 2) inhibition of AIV binding by this polypeptide (notably at a very high 10 molar concentration) may provide an indication of amino acid sequences that may contribute to ele-;l,usl~Lic interactions in the AT4 receptor binding site (e.g., RlIR2HR2, where Rl is an amino acid with an aromatic side chain such as OH, SH, or NH, and R2 is a polar amino acid).
The results of the tissue distribution studies, above, inrlic~te that the receptor is present in the adrenal tissues in most .. ,.. ~ n species, and can be isolated in P2 lllblal1e prep~lions from most m~mm~ n species.
Receptor Autoradio~raphy in Tissues:
Receptor autoradiography is a useful extension of radioligand binding studies since it provides detailed anatomical hlrul'''alion about the location of receptors in 20 tissues and groups of cells in tissues, and thus it f~rilit~tes underst~n-ling the function of the AIV ligands and AT4 receptors in those sites. Autoradiographic analyses of serial sections of guinea pig brain (20mM thirlrness) were pelro,llled. The autoradiographs showed a pattem of distribution for AII receptors and AT4 receptors in the H~hemll~ (Figure 3), Hippocampus (data not shown) Cerebellum (data not 25 shown), Plt;fiullli~l Cortex (data not shown), and Th~l~mlls (data not shown). In each case the receptor distribution in the tissue was detemlined by binding of 125I-Sarl,Ile8-AII, or 125I-AIV, re~,ec~ ely. Specificity of ligand binding in these autoradiographic studies was d~..oh~ led by co~ Jetil1g the binding of the specific ligand (i.e., AII or AIV) with l...l~hF.led 100nM Sarl,Ile8-AII, or 100nM AIV, 30 respectively. The data dellloh~lale that while specific AII and AT4 receptors are located at similar sites in the Habenula, Hippocampus, and Cerebellum of guinea pig brain, the two l~;c~l"ol~ are distinct with regard to exact groups of cells that express the two di~lenl receplol~. Major differences were observed in the ~I~;fiolllal Cortex and Th~l~mlls where AT4 receptors were abundant but AII receptors appear to be 3 5 relatively rare.

WO 94/00492 . ~ PCr/US93/0603~
?~.3g~0~ -42-In the Hippocampus the AT4 receptor is present in the pyramidal cell layer CA1, CA2, and CA3 of the Hippoc~mpus and dentate gyrus. Binding of AIV
occurred at a single binding site with high affinity (Kd=1.29_0.18nM, mean + SD, Hill Coef. = 0.993+0.015) and in a saturable manner (BmaX-449_62 fmol/mg protein).
5 The propel lies of the hippocampal AIV ligand-receptor system as described in greater detail below (see Fx~mple 7). The fintlines of AT4 receptol~ in the Hippocampus suggest that the AIV ligand-receptor interactions in the Hippocampus may me~i~teunique angiotensin-dependent functions in~ ing memory ~l~h~l~ce...el~l The AIV
ligand-receptor system may provide a link bt;~wc;en the Hippocampus and memory.
The mutually exclusive cellular distribution of AIV and AII receptors is de,llon~ ed in the autoradiograph shown in Figure 3. Panel A reveals intense 125I-AIV binding in the habenula, while Panel D inriic~tes that 125I-Sarl,Ile8-AII
binding is localized primarily to fiber tracts inr.lu-ling the visual te~...r...l~l relay zone and the medial !~...,-i~c~.s. The specificity of ligand binding to receptors in these 15 tissues was illustrated by co...~ g 125I-AIV binding with lOOnM non-labeled AIV
(not AII) [(Figure 3, Panels B and C)]; and, lOOnM non-labeled Sarl,Ile8-AII
displaced only the 125I-Sarl,Ile8-AII binding [(Figure 3, Panels E and F)]. Sometissues, however, may contain both AII and AT4 receplo,~.
Q~ live aspects of binding in brain is presented in Fx~mple 7, and other 20 tissues data is presented in Example 1, above. The results show that all illlpolL~Il cardiovascular tissues in guinea pigs contain the AT4 receptor. This result is not surprising in light of the observation (above) that vascular endothelial cells contain high concentrations of lecep~ol~, but this is not re~,ollsible for tissue binding of AIV
ligand because every vascularized tissue will possess AT4 receptol~, i.e., skin and 25 skeletal muscle has low levels of lecep~or.
Materials and Methods:
Autoradiographic analysis of 125I-AIV and 125I-Sarl,Ile8-AlI binding in guinea pig tissue was dettlll~ed as follows. Heart, kidney, brain, and other tissues were cryostat-sectioned into 20mm section~ that were mounted on chrome-alum-30 gelatin-coated slides in mllltiple sets of seven. The slide-mounted tissue sections were thawed (35C) and pr~inr,ub~ted in assay buffer (150mM NaCl, 50mM Tris, 5mM
EDTA, 0.1% BSA, lO~M bestatin, 5011M Plummer's inhibitor, lOO~lM PMSF, at pH7.4) for 30 min. and then inr,ubated for 1 h in the same buffer with the addition of 225-250pM of 125I-Sarl,Ile8-AII (for viell~li7ing AII receptors) or 125I-AIV (for 35 vi~u~li7ing AT4 receptors). To define the specificity of the ligand binding, tissue sections were incub~ted in the radioligand in the presence and absence of lOOnM

`'VO 94/00492 2 1 3 9 1 0 5 PCr/US93/06038 unlabeled AII or AIV peptide. After appropliate washing, autoradiograms were prepared by apposing the slide-mounted tissue sections to X-ray film (Hyperfilm,Amersham) for an appl Opl iate exposure time. The amount of radioligand binding in a tissue was quantified using densitometric techniques and 125I standards (Microscales, 5 Amersham, Arlington Hts, IL).

Receptor Isolation~ Purification. and Pl opel ~ies and Production of Monoclonal Antibodies Receptor Isolation and Purification:
10The AT4 reccll~or was solubilized in high yield from purified bovine adrenal ~clllbl~1es using the zwitterionic dclergclll CHAPS (1%) at 4C over 4 h under conditions where peptidase activity and diffclclllial solubilization of the AT4 receptor (but not the AII receptors) is pelll,iLled (see also Example 4, Materials and Methods, below). For L-~...ple, lllcl~ es from a variety of di~cncn~ tissues and cells (e.g., 1525mg of P2 ",e",~ es, E~,lple 1) were inc~lb~ted for 4 h in Hepes buffer (20mM, pH7.4) co.~ g 1% CHAPS and a cocktail of protease inhibitors and alternative protease sub~ es, i.e., lO~lM bestatin; 50~1M Plummers' inhibitor; 0.2% BSA
(bovine serum albumin); and lOOIlM PMSF (phenylmethylsulfonyl fluoride).
A most useful collll)oncllt of any AII receptor purification scheme was 20 in~ (ling a step whcrein the solubilized ~lclll~ e proteins were subjected to a heat L~ c~ l at 60C, e.g., for 20 min~ltes and in the plescllce of 20mM Mg++. This step was useful in dcsLluying any residual AII receptor leaving the AT4 receptor intact.
The AT4 receptor was stable to cl-ro"lalofocusing and SDS-PAGE, allowing isoelectric focusing, or one- or two-dimensional PAGE or SDS-PAGE to be used for25 purification. Due tû the slow-ûff rate of 125I-AIV binding, the receptor was radiolabeled with l25I-AIV ligand to allow ease of identification during purification.
As an additional aid to purification, the rccepLor was sllccçssfiJlly cross-linked to a 125I-radiolabeled AIV analog ligand having a C-terminal extension, i.e., from residue 8, with Iysine residues (i.e., l25I-Lysll-AIV). The Lysll-AIV analog binds to 30 the AT4 leceplor with a Kd that is similar to AIV ligand. Using Bis (sulfos~lcGinimi~lly) ~ubclilllidate (BS3) as the cross-linking agent, the 125I-Lysll-AIV
analog of AIV was bound to the AT4 receptor and then cross-linked to the AT4 eccp~or through the e-amino group of Lys. Purification of the AT4 receptor may also be achieved, for example, by ion exçh~nge, lectin cl~rolllaLography, hydrophobic 35 chrolllaLography with conventional techniques, HPLC, or FPLC.

- 21391o5 P~t/US93/0603 ~44~ 03 Rec'd PCT/PT0 15 JUl t~

SDS-PAGE analysis of isolated and purified receptor indicated a molecular weight between 130KDa and 150KDa, at about 146KDa for the BS3-cross-linked AT4 receptor from bovine adrenal tissue. The purified, uncrosslinked receptor appears to have a significantly smaller molecular weight, on the order of 60KDa.5 Receptor Properties:
Identification of the family to which a receptor belongs commonly permits predictions to be made about possible improvements in purification, useful methods for stabilizing the receptor during purification, cellular sources and assays useful for molecular cloning of the receptor, and identification of novel physiological roles for a 10 receptor. For in~t~nce, neulol~a~ rs and hormones are known to interact with four types of plasma "lt",b,~1e receptors: 1) mlllti~llbunit receptors that regulate an intrinsic ion ch~nn~l; 2) G-protein linked receptors that, via the G-protein, can activate membrane ch~nn~lc and enzymes; 3) guanylate cyclase receptors that possess intrinsic guanylate cyclase activity in a single membrane spanning polypeptide chain;
15 and, 4) protein tyrosine kinase receptors that have intrinsic tyrosine kinase activity capable of phosphorylating multiple protein substrates.
Many comm~n ne~,o~ rs like acetylcholine, glycine, glllt~m~te, and GABA activate receptor-ion çh~nn~lc. The interaction of the neulo~ i...;ller andreceptor results in the opening of an intrinsic ion ~h~nnPl In all cases these receptors 20 are constructed as heteromllltim~rs and are most likely evolutionarily related. Despite the h"~o"ance of this receptor class, to our knowledge no known peptide transmitter or hormone acts by such a ~"ech~ m Thus, it is reasoned unlikely that the AT4 receptor is a member of this family of receptors.
Studies have been conducted to determine the receptor family to which the 25 AT4 receptor belongs (see Examples 5 and 7). It has been reported previously that the AII receptor may be a member of the G-protein-linked family of cellular receptors.
The majority of known peptide receptors belonging to this family are characterized by seven membrane-spAnl-il-g alpha-helical regions and when stiml~l~ted are capable of activating membrane bound enzymes like adenylate cyclase, phosphodiesterase, and30 phospholipase C. (30). Additionally, membrane channel or ion transporter plope-lies can be indirectly modified by the intervening G-protein (31). Although many strategies have been devised to test a particular receptor's linkage with a G-protein, three strategies seem to predominate. In one form or another these include the following approaches: 1) GTP and its analogs are known to alter the binding affinity 35 of agonists to their receptors. Therefore, the ability of GTP or analogs to change agonist-binding affinity is diagnostic of a G-protein-linked receptor In the presence ~'S'.R~71~251,B DOC
AMENDED ~;HEET

~O 94/00492 2 1 3 9 1 0 5 PCI/US93/06038 of GTP, dissociation of the G-protein subunits from the receptor results in a lowered afflnity for agonists. This was PY~mined (see Example 5) by the direct ~esesemçnt of GTP (of GTP~S) effects on agonist binding via chal1ges in dissociation rates or total binding over a range of GTP conce~ alions~ or indirectly by monitoring shifts in ICso values for agonists during competition for antagonist binding. 2) Another indication of G-protein linkage is the ability of agonists to stim~ te the intrinsic GTPase activity of the alpha subunit of G-proteins. This GTPase activity is triggered following receptor occupation and subsequent dissociation of alpha and beta-gamma subunits.
3) A final approach is to determine whether an agonist can f~ t~te nucleotide cycling. A crucial step in G-protein signal tr~ned~lction is the agonist-stim.ll~te~l dissociation of GDP from the alpha-subunit and its repl~cPmPnt with GTP. Changesin cycling are often ~esessed by co"")a,ing the binding of radiolabeled irreversibly bound GTP analogs before and after agonist stiml-l~tiQn.
Studies to date include studies to d~Le""i"e the cellular signal tr~ned-lction ".e~ ."e activated following binding of AIV ligand to the receptor. The data obtained with ieol~ted AT4 receptor now ~L,ongly suggest that although the AT4 receptor may be G-protein linked in certain cells (see Example 5) the AT4 lecepLor does not belong to the çl~esic~l family of G-protein-linked receptors for at least three reasons: namely, 1.) Solubilization and stability characteristics of the AII receptor (i.e., binding 125I-Sarl,Ile8 AII) and the AT4 ~cepLo~ (i.e., binding 125I-AIV) are significantly di~elt;"~ which is consisLe~lL with: a) large structural di~le,lces between the two leceplo,~, and, b) dirrere"ces in the structural basis of receptor integration into ~"e~ es. Thus, it is re~eon~ble to assume that if the AIVATl receptor is a ",e",l)el of the G-protein linked family of receptors, then the AT4 receptor probably is not.
2.) Receplo,~ of the G-protein-linlced family of leCep~Ol~ are reportedly susceptible to inhibition at micromolar concenL.aLions of GTP~S. Studies were thelero~t; contl~1cte~ to ~Y~mine 125I-AIV ligand binding to the AT4 receptor in the presellce of GTPy S. The binding of radiolabeled AIV ligand to AT4 receptors isolated from bovine adrenal ",e",l"~ es is not altered by adding GTPy S to the assay buffer at conc~ lions ranging from 10-10M- lO~M. (Under these conditions binding of control pltpalaLions of AII ligand to AII receptors [i.e. in the sameme",b,~le pre~ Lions] revealed the typical pattern of a G-protein linked receptor with decreased binding of 125I-Sarl,Ile8 AII at i"creas;,-g conce"L~Lions of GTP~ S;
number of t;AI,t;,i",ents = 5).

WO 94/00492 PCr/US93/0603 2~,3g~1~5 -46-3.) The AT4 receptor has a demonstrated molecular size of 140 to 150kDa (on SDS-PAGE) for the isolated and purified receptor, and 146KDa for the BS3 cross-linked bovine adrenal AT4 receptor. These molecular sizes are significantly di~.t;lll from the molecular weights of 55KDa to 65KDa that are 5 commonly associated with members of the G-protein-linked family of receptors.
If the AIV site is not a classical G-protein-linked receptor, then to what family of receptors does it belong? Evidence in recent years indicates the presence of peptide receptors with intrinsic guanylate cyclase activity. These receptors, best exemplified by the .. ~.. ~li~n ANP receptor, consist of a single polypeptide chain 10 with one melllb~1e-sp~nning region that possesses guanylate cyclase activity that resides near the intr~cçll~ r C-terminus(32). Since only two such ~.-~---,-.~li~n receptors have been identified (to date), the ANP and rat ;~le~ enterotoxin receptor, it is difficult to speculate on the probability that the AT4 lecep~or is a Illelllber of such a family of receptors. Nevertheless, the similarity in the molecular 15 weights and in ion requirements of the ANP and AT4 receptors neces~ tes the consideration that the AT4 receptor may be a member of such a family.
The final receptor family to which the AT4 receptor may belong is the tyrosine-kinase growth factor family of recel~lo,~. These receptors are characterized by a protein kinase activity which p,e~elellLially phosphorylates tyrosine residues.
20 Among the substrates of phosphorylation are the leceplor itself and phospholipase C, which when phosphorylated initiates the inositol phosphate c~c~de (33). The tissue response to prototypical peptides which act as tyrosine kinase receptors in~hldes long-term alterations that invariably involve chal~ges in the l-~s~ lion rate of selective mRNAs. Although often accol.lpal~ied by acute effects, these peptides appear to play 25 a role in the adaptation of target tissues to chronic cl~nges in the level of a factor. In addition, tyrosine kinase receptors often "cross talk" with other cellular receptor types (34) in lesponse to physiological and cl-~...;c~l stimuli. This type of role is precisely the function envisaged for the AIV ligand-,c;ce~lor system.
A co.llp& ison of the solubilization, physical p.ope.lies, and functional 30 activities of the AT4 eceptor with the cellular biology of ebe.~ of the tyrosine kinase family of growth factor recepto. ~ (e.g., fibroblast growth factor . C;Ct;~lo., FGF) s~l~gestC a closer relationship of the AT4 receptor to this family of c;ce~to~ than to the guanylate cyclase family of receptors. For ;~ llc~ both the AT4 receptor andthe FGF receptor have related biochemical characteristics, e.g., the FGF receptor has a molecular weight of about 140-150kDa (35), is relatively heat stable (i.e., at 56C), and has divalent ion req~ elll~ s (28). Moreover, as described herein, AT4 `~0 94/00492 2 1 3 9 1 0 5 PCI/US93/06038 -receptors appear to have growth factor activity on at least endothelial cells and myocytes. (In the latter case, the tissue distribution and the activities of AT4receptors are also consistent with a role for AT4 receptors in growth regulation. For ;.,~l~nce, as disclosed above, high concentration of AT4 receptors is present in5 cardiovascular tissues where angiotensins are reported to enhance tissue growth.) At least three observations are significant in ~ccigning the AT4 receptor to a receptor family. First, the molecular weight of the AT4 receptor is in the range of .,.~...be. ~ of the tyrosine kinase families of receptors. Second, the AT4 receptor, like members of both the tyrosine kinase families of receplo-~, is characterized by divalent 10 cation binding sites (i.e., Mg++). And third, the AT4 receptor, like members of the tyrosine kinase and guanylate cyclase families of receptors, is characterized byrelatively high heat stability (i.e., 60C/20...;....les). (For colllpalisol1, the epidermal growth factor .ece~lor (EGF) is heat stable at 50C for 30 min., and has specific binding sites for Mn++ and Mg++ [28]). Thus, by at least these criteria the AT4 15 recep~or appears to be a ...e...bel of the tyrosine kinase farnily of receptors, and not the G-protein-linked family of leCePLO-~. EA~.;...t;..Lal approaches to validate this vision are presented below, the e,~t;-i-.-ents ~ e the ability of AIV ligand to stim~ te phosphorylation of tyrosine residues in ...~ e proteins. In addition, the focus of the e A~.i.--ents that follow in Example4 (below) was directed toward 20 ~lP.fining the cellular biology of the AIV ligand receptor interaction, and these studies will also help confirm the c1~c.cific~tion of the AT4 recep~or as a member of the tyrosine kinase family of receptor (e.g., capable of re~ ting cell growth and intrinsic tyrosine kinase activity of the AT4 1 eceptor).
Materials and Methods:
25 Cross-linking to the AT4 lece~tor Cross-linking 125I-AIV to the AT4 receptor can be accomplished with Bis (sulfosuccinimi~lyl) s~e-ill idate (BS3) as diccussed above. The cross-linked recepLol (approx. mw of 146,000) can then be electroeluted from PAGE gel slices in a s~s~ 11y pure form for use as an electrophoretic sL~1da.d.
For cross-linking one milligram of total solubilized .. e.. ~.~le protein col~ .;..g AT4 recep~or was il.-"~bAIed with 30 x lO~cpm of 125I-AIV in 50mM
Tris, pH7.4 and 150mM NaCI col-lAinil~g a cocktail of pro~casc/peptidase inhibitors for 2 hr at 37C (final volume 0.5ml). Following inr.~1bation, the in~bate was spun through two succeccive spin columns packed with 0.8ml of BiogelP-6 t;AL~ne 35 (Biorad) that has been pre-equilibrated with 20mM NaP buffer, pH7.4 co.~l~inil~g 0.01% CHAPS.

WO 94/00492 2~L3910S PCI/US93/0603 The labeled receptor, now in phosphate buffer, was cross-linked with BS3 (final conc. 9mM; added as 90mM in DMSO). The mixture was incubated 30 min. at 0C. Cross-linking was termin~ted by the addition of lOOml of lM Tris, pH9.0 with an additional 10 min. inr,~lb~tion at 0C. The mixture was then spun through a final spin column to remove reactant and free ligand. The centrifugate was now ready for PAGE.
Production of Monoclonal Antibodies:
Monoclonal antibodies are useful for purification of receptor, and for ide~lLirying the receptor (and fragmPntc thereof) in tissues, cells, and biological fluids.
Purified or semi-purified AT4 receptor (preferably nontl~n~hlred) can be used as an in vivo or in vitro immlmogen. (Those skilled artisans will recognize a variety of options available to them for evoking monoclonal and polyclonal antibodies, e.g., see Harlow, E. and D. Lane, Eds. "Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory, 1988). For in vitro immlmi7~tion antigen can be in~bated in picogram ql)~ntities with murine, rat, or human lymphocytes. Production of antibodies can be screened by testing for the ability of l25I-AIV-prelabeled receptor to bind to antibodies adsorbed on a poly~Lylci-e plastic surface, e.g., in 96 well plates;
or, alternatively, by testing the ability of the antibody to inhibit binding of a purified labeled receptor to AIV ligand adsorbed to a solid phase. In either case, antibody producing cells are idçntifiP,(l, cultured, and cloned. The monoclonal antibody product of the cloned cell lines can bind the AT4 receptor in ligand-binding and non-binding domains of the AT4 receptor. Non-binding domains can include structural regions of the molecule as well as enzyme active sites, phosphorylation sites, glycosylation sites, and the like. The presence of antibodies specific for the ligand-binding domain can be ~csessed directly via the ability of the mono to competitively inhibit in binding assays. As mPnfionP,d earlier antibodies are useful for receptor . purification and immllnohistochP.miG~l studies de-si~ed to Pl~lrid~te the cellular location of receptors and also in structure/activity studies decigned to map functional domains in the receptor.

AIV Receptor Antagonists and Agonists To test the ability of ~y~ P~;~ed AIV ligands to co,llpcLiLi~ely inhibit for 125I-AIV ligand binding to the AT4 lecep~or, displ~cPmPnt curves were constructed using heat-treated (60C for 20 min. in 20mM MgC12) purified bovine adrenal cortical mel"l"anes using Methods desc,ibed in Example 1, above. Effects of AIV analogueson renal blood flow were determined as described in Example 6.

~vo 94/00492 PCl /US93/06038 49~13~1Q5 The design of AIV analogs followed a question based approach. The unifying question: What are the essçnti~l ligand domains for receptor binding and activation?
Individual chemical modifications were made to ask specific questions about spacial orientation of molecular surfaces, charge, hydrophobicity and occupancy of space5 (volume occupied at a specific location). A standardized assay of analog competition was employed to study receptor-ligand binding affinity of the high affinity l25I-AIV-binding receptor in heat treated, sucrose density gradient purified bovine adrenal cortical ",tl"b,~les. Energy ..~ ed7 computer generated models ("Macromodel"
program run on a Vax ",a"~fi~le) provided the visual representations of the 10 molecular col~""alion of highest probability.
Agonist versus antagonist activity was ~csesced using a laser doppler to monitor renal cortical blood flow f~llowing infusion of a test analog into the renal artery (see F.Yh~llple 6). Maximal response was co",pa,ed to the le~l,ollse (increased in flow) to AIV. (Note that under these conditions AII produces a decrease in blood 15 flow in this assay.) Inte,,ule~lalion of physiologic and binding data was based on the precept of a lock and key model of receptor binding and that dynamic change of the receptor upon interaction of the ligand was required for activation (full agonist activity; with second m~s~el~eel activation).
The following main assumptions were used:
1.) T.ig~nds with the highest affinity, when modeled in an energy .. ~ ed co~ru""alion offer a visual ,~ s~ hl;on of the receptor binding site field surface (i.e., hydrophobic charge interactions) and charge locations in the "pre-binding state"
and "non-activated state" (i.e., just as a clay imprint on a well fitting key represents the interaction surface of the lock).
2.) Specific ligand domains induce çh~l~g~o~ in the receptor upon binding that produce cellular responses. T.ig~n~s that fit the "pre-binding" receptor with high affinity may not activate the receptor, i.e., they may act as antagonists, whilestructures that induce ~h~n~,cs in the collrullllalion ofthe receptor may be cor"palille with, and part of, the cl-AI-g~s that produce high affinity binding, i.e., they may act as agonists.
The following is a ~ullllllaly of the questions asked and the compounds ~y..ll.~ci~ed to identify ~n~gonictc and agonists of the AT4 receptor that interfer with binding of physiological AIV fr~gm~ntc. The inventors believe that mapping the recel)lor binding site (herein) and underst~n~ling of the structure of the receptor and 35 its signal tr~nc~llction meçh~nicm form the requisite basis for rational design of WO 94/00492 PCr/US93/06038 2139105 ~50-pharmacologic therapeutic agents that interact with this receptor system in vivo in ",~""..~
Question #1. What are the absolute AIV ligand amino acid requirements for binding to the AT4 receptor?
The approach used to answer this question involved deletion of residues from either the N- or C-termius of AIV (i.e., VYIHPF), or from the larger AI sequence of which AIV is a part (i.e., Figure 1). For the most part these studies employed the bovine adrenal cortical AT4 receptor present in melllbl~i1e p-epa-~lions pl~pared as described above in Example 1. The binding assays were also cond~lcted as described in Examples 1 and 2, above, and certain of the results summarized here are also presented above in those E~llples.
Deletion of the N-terminal Vall residue from VYIHPF to produce YI~PF
reduced binding affinity to the bovine adrenal cortical AT4 receptor by 1000-fold.
Addition of d-arginine to the N-terminal, (i.e., a peptidase analogue of AIII), reduced affinity by 100-fold. Deletion of the C-terminal Phe (i.e., des-Phe6AIV) did not alter binding signifis~ntly. Further truncation of the C-terminal Pro5, however, produced a moderate affinity (i.e., 21-fold less than AIV). FragsnP-nt~ co~ g less than positions #l through #4 (i.e., N-VYIH-C) have Kj's>500nM. Addition of hi~titline to the C-terminus, (i.e., AI(3 9~, Figure 1), did not alter binding ~ignifir~ntly, and further addition of leucine (i.e., AI(3 10) ~igure 1) reduced affinity by just 2-fold and resulted in data best plotted in Scalcha~d analysis to fit a two site binding model.
These results suggest that the binding domain in the AT4 receptor recognizes the N-terminus of AIV with a high degree of specificity. The receptor appears tointeract less closely with the C-terminal region of AIV, but binding of receptors to this region of AIV may determine the receptor subtype specificity of AT4 receptors indi~elelll tissues.
Ouestion #2. Does the binding site that interacts with the #1 residue in AIV
(i.e., valine) exhibit any ~leleo~lecilicity for particular orientations of the N-terminal residue?
Repl~cP-mPnt of the L-valinel in AIV with D-valinel reduced binding affinity by 1000-fold. This in~lic~tçs that the domain in the AT4 receptor binding site thatinteracts with the #1 position amino acid residue in AIV possesses a minimllm of "4 non-planar ligand interacting sub-domains that have a fixed spacial orientation" that can be dç~ign~ted by the L-cGl~llllaLion of an L-valine amino acid. Examples of the latter "4 non-planar ligand interacting sub-domains" may be supplied by the side chain residues of 4 amino acids that appear in a requisite 3-dimensional space within this '~'0 94/00492 2 1 3 9 1 0 5 PCI/US93/06038 subdomain of the receptor binding site. (Results ~iccllcced in response to Question #4, suggest that one of the 4 non-planar ligand interacting sub-domains interacts with the 1-amine in the N-terminal amino acid.) Compounds that mimic the space filled by L-valine in a hydrophobic en~holll"~ may mimic the interactions of L-valine with 5 this subdomain of the receptor.
Question #3 . Is the hydrophobic nature of the Rl-group (i.e., Vall) in AIV a requirement for receptor binding and agonist activity?
Four analogues were synth~ci7ed and tested. Substitution of Vall with Ilel produced a slightly more hydrophobic peptide (i.e., IYIHPF) as determined by 10 retention on reverse phase HPLC, and this peptide exhibited a slight increase in binding to AT4 It;C~;~)L(jl~. Substitution of Vall with Phe greatly increased hydrophobicity but decreased binding affinity to the AT4 receptor by 4-fold.
SullJIisingly, substitution of Vall with Lys (i.e., KYIHPF) co..lA;..;..g a positively charged side chain, greatly de~ileased hydrophobicity but hlcleased binding affinity to 15 the receptor by more than 45-fold. Sul.slilulion of Vall with a negatively ch~,ed side chain (i.e., Asp) resulted in an analogue (i.e., DYI~F) with virtually no affinity for the AT4 receptor.
These results indiçate that the nature of the Rl-group (i.e., a rigid aromatic ring versus a flexible ~liph~sic carbon chain having an optional positive charge) 20 dictates the interaction with the binding site in the AT4 receptor, and not the just the degree of hydrophobicity of the amino acid residue. The results presented in Figures 5A and 5B also in~icate that Lysl-AIV (i.e., KYI~F) exhibits full (or increased) agonist activity relative to AIV (i.e., VYIHPF). Figure 5 shows changes in blood flow that result from binding of agonist Lysl-AIV (i.e., KYIHPF) to AT4 25 receptors in kidney, without ~h~ges in systemic blood p,es~ule. Systemic arterial pressure and cortical renal blood flow were measured as desc,ibed in Example 3, above. (No. of e,.~;,i",enls= 10.) Figure 5A shows no ci nific~nt chA.~ges in arterial blood pressure following a~h~ cllation of KYI~F at lOOpmole/25mVmin (open circles) or saline control (closed circles). Figure 5B shows çhAl~ges in renal blood 30 flow following a~l...;..~.alion of KYI~F at lOOpmole/25ml/min (open circles) or saline control (closed circles), with the increased blood flow being equal to 38% of the ~ x;~ a~ hle with a strong vAco-lilAtory agent (i.e., bradykinin, as described above).
Question #4. Does the ,ulilll~y (1)amine in the N-terminal amino acid 35 interact specifically with the Vall-binding subdomain in the AT4 receptor binding site?

WO 94/00492 ~L39~n 52 PCr/US93/0603R

As described above in response to Question#2, IlelYIHPF binds to the receptor with nearly the same binding affinity as VYIHPF. Methylation of Ilel in the latter peptide (i.e., to form N-methyl-IlelYIHPF) reduced bindng affinity for the AT4 receptor by 67-fold. Substitution of a secondary amine into the R1 position of AIV
5 (i.e., ProlYIHPF) reduced binding affinity to the AT4 receptor by 8-fold.
Substitution of R1 with benzoic acid (a partial structural analogue of Phe) or with

6-amino hexanoic acid (a structural analog of Lys) produced peptides with Ki's >lmM. Pl~cçm~nt of GABA (gamma-amino-butyric acid) in the R1 position decreased binding by 250-fold, i.e., relative to binding with AIV.
This data sllggtoctc that the lece~.lor contains a binding site sub-domain that closely interacts with the p~hllaly amine function in the R1 residue with respect to absolute space oci~p~ncy (volume) and probably a eleillosL~Lic charge, i.e., thereceptor non-planar NH3-binding colllpon~llL of the R1-binding sub-domain (the same non-planar sub-domain component described in response to Question #1 above), most likely is a negatively charged residue that resides adjacent to the 1-amine when the Rl group is ç~in~ the receptor sub-domain.
Question #5. Is the positive charge of the e-amine in Lys1 responsible for the increased binding affinity of KYIHPF to the AT4 receptor, or is this plopt;lLy attributable to the flexible, linear carbon chain?
Four di~e~ellL Rl positicn AIV analogues were synth~ci7ed to answer this question: 1) Lysl-substituted AIV (i.e., KYIHPF); 2) norleucine-substituted AIV,(i.e., NLel-YIHPF); 3) ornithine-substituted AIV (Ornl-YIHPF); and, 4) norvaline-substitubed AIV (i.e., Nval-YIHPF). The çh~mic~l structures of these side chains are shown in Table 10.

Chemical Structures of Aliphatic Carbon Side Chains Lys Nle Orn Nv CH2 l H2 l 2 l H2 l H2 l H2 l 2 CH3 '~'0 94/00492 21 39 1 05 PCI/US93/06038 NVa-substituted AIV had a 4-fold greater afflnity for the AT4 receptor than Orn-substituted AIV. Nle-substituted AIV had a remarkable binding afflnity 60-fold higher than Lys-substituted AIV: i.e., NlelYIHPF had a Ki of <1 x 10-12M, a virtually irreversible binding ligand and indicative of partial-agonist activity. To confirm the 5 agonist activity of Nle-substituted AIV, studies were con~lcted to evaluate the ability of this analogue to stim~ te m~im~l arterial blood flow in rat renal arteries. The studies were con.1ucted as described Example 6, above. Infusion of 0.10 picomoles/minute of NlelYIHPF into the rat renal artery produced the effect of lll~imal blood flow, however, the absolute levels of flow stimlll~ted by this analogue 10 were less than the absolute levels produced by AIV or LyslYIHPF, intlic~ting that NlelYIHPF is a partial agonist Figures 6A and 6B, described below. Figure 6A
shows ~.h~nges in arterial blood pressure following ad...;l.xLl~Lion of NorLeuYIHPF at 1 OOpmole/25mUmin (open circles), 50pmole/25ml/min (open squares) or saline - control (closed squares). Figure 6B shows çh~nges in renal blood flow following a~minetration of NorLeuYIHPF at 100pmole/25ml/min (open circles), 50pmole/25ml/min (open squares) or saline control (closed squares). The infusion of 0.05pmole NorLeulYIHPF had no effect on mean arterial pressure (Figure 6A) but increased renal blood flow in a dose-dependent lll~mCl. a m~imum of 19% increasein renal blood flow was observed with infusions of 0.05pmole (Figure 6B); 19% also at 0.1 pmole (Figure 6B); 21% at 10pmole (Figure 6B); and, 100pmole NorLeulYIHPF increased renal blood flow by 30% (Figure 6B). (Infusion of 0.15M
NaCl in control animals were without any .eignifi~.~nt effect.) The data in-licates that a flexible, linear carbon chain interacts specifically with the receptor in a high affinity ",al~nc" chains having a four carbon atoms bind with a higher affinity than chains with three carbon atoms; a positive charge is deleterious to binding, but does provide an analogue having full agonist activity (i.e., Nlel-AIV).
Ouestion #6. What is the specificity ofthe lecepLor for the R2 residue?
Analogues were plc~arcd with ~tyrosine s~lbstit~ltion for L-tyrosine in the R2 position of AIV (i.e., D-Tyr2 AIV). The latter analogues exhibited low binding affinity for the AT4 rcceptor. Reversal of the positions of the Phe and Tyr residues in AIV (i.e., Phe2Tyr6 AIV; VPIHYF) also resulted in analogues that had very low binding affinity.
These results suggest strict recognition of the R2 Tyr residue, possibly throughhydrophobic and hydrogen-bonding interactions. Substitution with Phe, Ala, and beta-alanine is useful to map the nature of the interactions with this sub-domain of the AT4 receptor binding site.

2139~5 ~54~

Question #7. Will the receptor tolerate the introduction of non-peptide bonds?
Compounds were synthP~i7ed with methylene bond isosteres (i.e., (-CH2-NH-) to answer this question. The synthesis was accomplished using the r~cem~te free amino aldehyde synthesis, Schiffs base formation, and reduction with sodium cyanoborohydride. Specifically, synthesis of +H3N-Val(CH2NH)Tyr-Val(CH2NH)-His-Pro-Phe-COO~ (design~ted divalinal AIV) was accomplished ~1tili7:ing standard solid phase protocols with t-Boc protected amino acids and amino aldehydes. The same general protocol is used to produce other AIV ligands with methylene bonds between desired amino acid residues using the applopliate amino acid aldehyde as a reagent. R-group protection was: Tosyl for His and 2,6-dichlorobenzyl for Tyr.
Synthesis occurred on a t-Boc-Phe s~sLiLuLed resin (0.76mmol/gram of 1% cross-linked divinyl ben,.ene resin from P~ninc~
For amino acid coupling the following protocol was used: methylene chloride wash: lX1 min; 45% w/v trifluoroacetic acid and 0.08% indole in methylene chloride deprotection: lX3 min and lX30 min; methylene chloride wash: 5X1 min;
isopropanol wash: 3Xl min; methylene chloride wash: 3Xl min; 10% v/v triethylamine in methylene chloride neutralization: lXl min and lX5 min; methylene chloride wash: 2Xl min; isoplopanol wash: 2X1 min; methylene chloride wash:
2X1 min; isopropanol wash: 2X1 min; methylene chloride wash: 3X1 min; amino acid coupling with a 2.5 or 5-fold excess of amino acid and EDC in methylene chloride: reaction times of 1.5 to 3.5 hours; methylene chloride wash: 3X1 min;
isopropal1ol wash: 3Xl min; methylene chloride wash: 3X1 min. The above protocol was repeated for each cycle. Re-links of amino acids repeated all stepsbegil-l-;l-g with the neutralization. All linkages and deprotections were monitored with the Kaiser ninhydrin test. Acylations less than 94% were repeated.
Valinal (N-t-Boc-L valine aldehyde from p~.nin~ ) was linked to the free amino-tenninal of the growing peptide by formation of a Schiffs base intermedi~te with subsequent bond reduction. For this reaction the above protocol was utilized with the following alterations: prior to coupling, the resin was washed with dimethyl Çc,l.."....;de 3Xl min; a 5-fold excess of valinal was added in 1% acetic acid/dimethyl ru,...~...;~e; a 10-fold mole ratio excess of sodium cynoborohydride (Sigma) wasdissolved in 3ml 1% acetic acid/dhll~Lhyl rol...i~..;de and added in equal aliquots at 0,3,5,10,15,20,25,30,40 and 50 min with concurrent nitrogen purge; the coupling was 35 allowed to continue for 70 additional min; the resin was washed with dimethyl wo 94/00492 2 1 3 9 1 0 5 PCI/US93/06038 _ follllalllide 3X1 min. Linkage was ~csessed with the Kaiser test and revealed a slightly reddish color of the beads when greater than 94%.
The fini~hrd N-terminal deprotected resin-linked peptide was cleaved from the resin and side chain deprotected with anhydrous HF co,~ g 10% anisole at 0C
for 40 min. The HF and anisole were removed under vacuum and the peptide washed with anhydrous ether. The peptide was extracted with 20% glacial acetic acid andIyophilized. The crude peptide was then purified by prepal~ e reversed phase HPLC
in two steps, the first an isocratic method using acetonitrile:triethylamine-phosphate, pH3 followed by a second gradient method using acr~ollillile:water (0.1% TFA). The purified product was analyzed by analytical reversed phase HPLC
(acetonitrile:triethylamine-phosphate, pH3) gradient method (12-18% over 60 min at 2ml/min).
Repl~c~m~nt of the Rl-R2 peptide bond with the methylene bond reduced affinity of binding to the AT4 receptor by 5-fold. Double repl~cçm~.nt of both the Rl-R2 and the R3-R4 peptide bonds and substitution of the R3 Val with Ile produced the peptide: N-Vl-CH2-NH-Y2V3-CH2-NH-H4P5F6-C (Divalinal AIV) that had equal or better affinity than AIV for the AT4 receptor. In addition, divalinal AIV has been shown to exhibit çnh~nced metabolic stability and to be a potent antagonist of AT4 receptor activity. Figure 11 illustrates the colllp~ e stability of 125I-AIV and 125I-Dival AIV following exposure to a lllt;~ e fraction prepaled from rat kidney.
Kidney was chosen as the tissue of study because of its well-known degradative capacity. The metabolish of l25I-AIV and 125I-Dival AIV by rat kidney ,llelll~ es was deLellllined as follows: Rat lllclllbl~es (25~g protein) were inr,ub~ted with .6nM
125I-peptide at room telll~lt;lalult; in a buffer co~ l-g Tris, 50mM, pH7.4; NaCl, 150mM; BSA, 0.1%; EDTA, 5mM; bestatin, 20~1M; and Plullllllrl's inhibitor, 50~M.Metabolism was stopped by the addition of acetonitrile (final concenLlalion 50%), and the s~mples were analyzed by reverse phase (Cl8) HPLC. As can be seen in Figure 11, AIV is rapidly degraded while Dival AIV remains 100% intact after 4 hr of inr,llb~tion.
In addition, following the procedures of E~l,ples 4 and 6, it has been found that preinfusion with divalinal AIV co...i.letkly blocks LyslAIV-in~uced increases in blood flow, and preinfusion with divalinal AIV actually ll~lSr~llll5 AIV's effects on blood flow from an increase to a decrease. This effect of divalinal AIV on AIV
sll~ests that AIV also acts at AII receptors, the effects of which are normally m~c~ed 35 by AIV's action on AT4 receptors. Divalinal AIV llr.~l,.lk,.l by itself did not alter W094/00492 21391S PCr/US93/0603 blood pressure or renal blood flow (Figure 12A). Additionally, it had no effect on AIV-ind~lced decreases in blood flow (Figure 12B).
It has been further found that AIV potenti~tes the pclroll,lance of rats in a passive avoidance task in a dose-dependent manner while AII exhibited no specific effect. In this experiment, the mean latency (see + SEM) for independent groups of rats to reenter the dark co~"?a"",ent following passive avoidance conditioning on Day 1. On Day 1 (5 min prior to testing for retention) the Control Group received 2~11 aCSF, angiotensinII (AII), AIV, or divalinal AIV. Each group except the divalinal AIV revealed ci nifiç~nt elevations in latency to reenter the dark colll?~",cll~ - co",?~hlg Days 1 and2. In addition, the groups that received 100pmole or lnmole of AIV indicated a signifiç~nt elevation in latency to reenter com?ared with those groups that received aCSF and AII, while these latter groups did not differ from each other. Rats treated with divalinal AIV were not st~tictic.~lly dirrclclll from preshock controls. I~llerc~lingly, Ire~l",~ of rats with divalinol AIV
blocked the typical increase in latency seen in control rats. Re~,onses by rats treated with divalinal AIV were not st~tictic~lly dirrc,c"~ than preshock controls. These data indicate that while AIV potently çnh~nced cognitive function, divalinal AIV acting as an AIV antagonist completely blocks the learning and/or retrieval of the passiveavoidance task. Fu~Lhc~ ole, these data suggest that endogenous AIV must play a critical role in cognitive function.
These results intlic.~te that the AT4 receptor binding site domain binds analogues in which the peptide bond has been replaced with a non-carbonyl (non-peptidase sensitive) bond that has a similar bond length, and that is non-planar and has a non-rigid carbon-nitrogen bond. Non-peptide bonds offer pharmacological advantages for a therapeutic composition, i.e., prolonged half-life. `
Ouestion #8. What delclll,ines agonist versus antgonist activity?
Both AIV (i.e., VYI~F) and Lysl-AIV (i.e., KYI~F) exhibit full agnoistic activity, while Mel-AIV (i.e., NleYI~F) is only a partial ~gonict The model capable of ~Ypl~ining this behavior has the following co",?onc"l parts:
a) The ,ecc~Lor binding site sub-domain interactions with the side groups (i.e., of Rl) determines receptor activation;
b) The interaction at the Rl-sub-domain binding site involves a hydrophobic pocket;
c) The space in the latter Lydlo?hobic pocket col~lllls very closely with the 4 carbon side chain of norleucine;

~'O 94/00492 2 1 3 9 1 0 ~ PCI/US93/06038 -57-~ ~

d) Mel (i.e., in NlelYI~F) interacts with the hydrophobic pocket without çh~nging the co~lll.alion of the pocket;
e) Vall (i.e., in VYI~F) must occupy an "e~p~nded"
hydrophobic pocket, i.e., where the receptor hydrophobic pocket is displaced laterally to accomodate the branched carbon side chain in these residues. Lysl (i.e., in KYIE~F) must similarly occupy an "exr~nded" hydrophobic pocket because of the charge repulsion from the hydrophobic "walls" of the pocket; and, f) The process of "c~ n~ g" the hydrophobic pocket con~titute~ a molecular trigger for the process transitioning the receptor from the "pre-binding state" to the "binding state".
To study the plopclLies of the "hydrophobic pocket" subdomain of the AT4 receptor binding site it is useful to prepare derivatives of Ornl (i.e., Ornl YI~F) at the delta amino group to: a) the charge of the group; b) place a planar, 15 col~llllaLionally-fixed bond in the 4 carbon side-chain group that will inhibit binding in the hydrophobic pocket if the "walls" of the pocket are unable to move to accomodate the space required by the col~lll.aLion; and, c) synthesi7e col~,...alionally-fixed bonds in carbon side-chains of di~.cnl length (e.g., 3-5carbons) to explore the optimal longihl-lin~l dimensions of the flexible wall space in 20 the receptor pocket. Suitable N-delta groups for this exploration are acetate, propionate, benzoic acid, isobutyric acid, and LlilllcLllyl acetic acid.
Question #9. Can the shorter peptide AIV(~ 1) analogues (e.g., VYIH) be converted to high affinity ligand by norleucine substitution at position Rl?
Answers to this question provide tetrapeptides agonists and antagonists whose 25 interactions with the AT4 receptor are easier to molecularly model, and mimic. The peptides Nlel-AIV(1 5) (i.e., MeYI~), Nlel-AIV(1 4) (i.e., NleYIH), and Nlel-AIV(1 3)(i.e., MeYI) may be useful for testing space-filling modifications that can be made to alter binding in the receptor binding site sub-dom~in~. It is considered highly likely that independent mo~lifir.~tions that can be made to alter the binding of 30 the latter small Nlel peptides into the AT4 cceptor binding site sub-domains will be paralleled when the modifir~tion are inco.l.o.~Led into larger AIV ligands.
Question #10. Will ~ub~LiLuLion of Ilel at position R6 (e.g., to form VYIHPI, KYIHPI, or NleYIHPI) create antagonist activity?
Three Ile6 substituted AIV analogues were synth~i7ed (VallIle6-AIV, 35 LyslIle6-AIV and NlelIle6-AIV). When tested for in vitro receptor binding activity VallIle6-AIV had a hi~her binding affinity for the AT4 receptor than AIV (i.e., WO 94/00492 PCr/US93/0603 2~.'3g~'j -58-VYIHPI >VYIHPF); and LyslIle6-AIV had a lower affinity than Lysl-AIV (i.e., K~IHPI <KYIHPF).
The results suggest that the AT4 receptor binding site is a multi-domain binding site with interactions such that binding in one sub-domain (e.g., within the S hydrophobic pocket of the Rl sub-domain) can be excluded by high affinity binding at a distant sub-domain site (e.g., within the subdomain with specificity for the C-terminal Ile6 or Pro5 residues; i.e., at the R6 subdomain binding site in the receptor). The in~luced-fit model supplied above in response to Question #8 is coll,palible with the observed exclusionary binding properties: i.e., binding of Rl 10 hydrophobic pocket that con~tit ltes the Rl-binding subdomain requires flexibility of expansion in the pocket, and binding of R6 in the R6 sub-domain binding site confers a rigidity to the ,eceplor that inhibits flexibility in the Rl-binding subdomain.
Materials and Methods: -Binding was carried out as described in Example 1, above, in siliconized glass culture tubes co.. ~ ing 0.2nM 125I-AIV, 25~1g of melllbl~le protein, and the desired analogue over a concellL-~Iion range of 10-12 to 10 4M using half-log dilutions. All binding inr,ub~tion~ were carried out in duplicate at 37C for 2 h in a buffer co~ g: 50mM Tris, lSOmM NaCl, 5mM EDTA, lO~,lM bestatin, 5011M
Plummer's l?e~gf-nt, 100~,1M PMSF and 2% BSA (Assay buffer) in a total volume of20 0.25ml. After incub~tion, the incub~tiQn ~ lures were filtered through glass fiber (GF-B) filters soaked in 0.3% polyethylf ~ -e and washed with 4-4ml washes of PBS. The filters were then counted on a Bec.~m~n 5500 gamma counter. A typical CA~I~lilllelll f .~..,;ned 5 analogues simlllt~neously and inrluded a positive control curve in which non-radiolabeled AIV ligand was used as the displacer to inhibit 25 binding of l25I-AIV to the AT4 receptor. All s~mrlf s were run in quadruplicate, each with a di~lenl tissue ~,e~)a,alion. Data was analyzed by the LIGAND program (29)from which Ki values were obtained. AIV analogues that are peptides were sy.lll-f~;~ed by the standard Merrifield method utili7.ing t-Boc protected amino acids and chloro",t;ll,ylated resins on a Vega 250 coupler automated Sy~ f"~ (as 30 des~i,il,ed in E~"ple 1, above). Following sy"lllesis, the crude peptides were purified by l)rel~ali~le reverse-phase HPLC. The amino acid co",;)osilion of thepurified peptides was dete"llll ed with respect to both co~ )osilion and total purity.
Typically the peptides used in these studies were greater than 99% pure and colllail ed about 20-25% acetate.

~ !0 94/00492 2 1 3 9 1 0 5 PCI /US93/06038 Vascular Effects of the AIV Ligand-AIV Receptor Interactions In endothelial cells (such as bovine colon~y venular endothelial cells), it has been reported previously that these cells may play a critical role in angiogenesis 5 (review, 21). In one study by others angiotensins were reported to be capable of sfim~ ting angiogenesis (22). However, studies in the inventors' laboratory over the past ten years have failed no less than six times to de,llollsllale detect~ble levels of AII receptors in prepal~lions of endothelial cells that were free of smooth muscle co..l;....;l~AI;on (a finding contradictory to one report that AII receptors may be present on endothelial cells, (23). In addition, AII and Sarl,Ile8-AII have beenreported to stim~ te bovine endothelial cell proliferation (24), but the possible mecl~ were not clear, especially in light of other studies reportedly showillg that AII and Sarl,Ile8-AlI were rapidly metabolized in tissues and biological fluids to smaller metabolites. In light of the present disclosure it is now clear, in hintl~ight that hydrolysis of AII or Am to AIV can result in binding of AIV to AT4 l~cep~ol~ on endothelial cells with triggering of cell proliferation, and may possibly be involved in the initiation of hyperplastic growth of endothelial cells or vascular smooth muscle cells.
AT4 l~ce~lolY, in vascular cells The following study describes the characteristics of a new class of angiotensin binding sites in vascular endothelium that exhibit high spe~.ifi~ity and affinity for h~;A~pe~ide AIV. Analysis of 125I-AIV binding was pelrolllled in melllbl~e fractions of two endothelial cell lines, bovine corol1~y venular endothelial cells (CVEC) and bovine aortic endothelial cells (BAEC). Kinetic analysis of binding 25 indicated that equilibrium was reached in 60 min. at 37C, le~Ai.-~d stable for at least 4 h, and produced a ç~lcl-lPted kinetic Kd Of 0.3nM. Saturation equilibrium binding studies analyzed by non-linear curve fitting s~lgge.~ted the following two site models (mean +/- SEM): CVEC Kdl=14.6 +/- 26.5pM, BmaXl = 6 +/- lfmol/mg protein, Kd2=4.4 +/- 0.8nM, BmaX2 = 434 +/- 51 fmol/mg protein. Compteition binding curves from CVEC d~.lol~l-a~ed high spe~.ifi~.ity of the recel)lor for for AIV. The colnl)t;lili~e binding affinities of analogues to the receptor showed ~ffinites that (in decreasing order) were AIV >AII(3 7~ >AIII >AII ~SarlIle8-AlI, or AII(4 8~
>>DUP 753, or CGP42112A. The AT4 receptor in endothelial cells may not be G-protein linked because the non-hydrolyzable GTP analog GTP~S had no effect on l25I-AIV binding to receptors in BAEC cells. These data indicate that AIV binds to a WO 94/00492 2~39~05 PCI/US93/0603 site in vascular tissues that is distinct and separate from the classic ATl or AT2 angiotensin receptors.
Kinetic binding studies Kinetic analysis of 125I-AIV binding to AT4 receptols in CVEC membrane 5 revealed that equilibirum was reached in approAhllalely 60 min. and rem~in~d stable for at least 4 h. at 37C (Figure 7A). The kinetic propclLies of 125I-AIV binding to AT4 receptors in Illt~ lane fractions of bovine colonaly venular endothelial cells (CVEC) at 37C. are shown in Figures 7A and 7B. The association (Figure 7A) and dissociation (Figure 7B) rate cons~ s for binding of 0.6nM l25I-AIV were 9.3 x 107 10 M~lmin~l and 0.028 min~l, respe~ ely. The kinetic Kd calculated from these rate con.cl~..ls is 0.3nM. C~lc~ tions were pelrulllled by the LIGAND program from a mean of four cAI,elilllenls with d~lp!icate samples. Data presented here represent the results from a single eApelill~ L.
Analysis of a~soci~tion data under pseudo first order rate conditions resulted in an observed association rat COIIS~ (kobs)= 0.084 +/- 0.013. The dissociation rate constatnt (k 1)= 0.028 +/- 0.005 min~1 was estim~ted by the addition of lmM
undlabeled ligand following inc~lb~tion of 0.6nM 125I-AIV for 120 min at 37 (Figure7B) and the actual association rate constant (k1) was calculated to be 9.3 x 107 M~lmin~l. Based on these rate COllS~all~S, the appa~en~ kinetic Kd value is 0.3nM. Less than 10% degradation of the ligand occurred under the binding conditions used here.
Equilibrium bindin~ studies As shown in Figures 7A and 7B, quilibirum binding of l25I-AIV to AT4 receptors in CVEC and BAEC Ill~lllbl~es reached saturation at 37C in 120 min.
Equilibrium saturation binding and ScaLcllald ~l~lsrulllla~ion analysis (insert) for 125I-AIV binding to AT4 recel)~olS in CVEC is shown in Figure 7A, and BAEC in Figure 7B, ll~cllll~l~le fractions after 12û min. at 37C. The data were best fit by a two site model ~ltili~in~ the non-linear curve fitting program LIGAND (No. of expts.=4, each with ~lllpli~te salllple~).
Sca~cllald ~l~lsrullllalion of these data sl~g~ested the presence of multiple binding sites in endothelial cell Ill~,.ll~l~le-associated AT4 receptors. The data were best resolved into two collll)oneellls collt;~l,ol1ding to a high and a low affinity binding site. The final Kd and BmaX values were as follows:
In CVEV (Figure 7A): site #1 14.6 +/- 26.5pM with 6 +/- lfmoVmg protein;
site #2 1.4 +/- 0.2nM with 594 +/- 4fmollmg protein; and, "'O 94/00492 2 13 9 1 0 5 PCl/US93/06038 In BAEC (Figure 7B): site #1 26.9 +/- 9pM with 10 +/- 2fmol/mg protein;
site #2 4.4 +/- 0.8nM with 434 +/- 51fmol/mg protein.
Values obtained when fitting the data to a single receptor affinity site model were: in CVEC 0.7 +/- O.lnM Kd with BmaX=lo +/- 2fmol/mg protein; and in BAEC
1.0 +/- 0.2nM with Bma,C-260 +/- 38fmol/mg protein. The two site model produced a significantly better fit for both cell types as compared with the single site model (i.e., F-test, p<0.001).
Co~llpclilion bindin~ studies C~n")elilion studies were con~lcted to rlicFl~ce 125I-AIV binding to AT4 receptors in CVEC membrane plepalalions with specific ligand, i.e., AIV, and other related angiotensin fr~grnP.ntc. Figure 8 shows co",pclilion displ~m~nt curves dPli~ ;ng the ability of angiotensin fr~gmPntc to inhibit specific binding of 0.5nM
125I-AIV to AT4 receptors in "lel,.b.~e prcpa.~lions of bovine coronaly venular endothelial cells (CVEC). (No. of exper. =2; each con~llcted with duplicate samples.) The rank order affinity of co.. pt;lili~e analogues con.l)clili~/ely displacing bound AIV from its receptor were as follows: AIV >AII(3 7) > AIII >AII
>SarlIle8-AII, or AII(48~ >>DUP753, or CGP42112A. (For sequences of AII
analogues see Figure 1). The results of these studies are su------a,i~ed in Table 11.

Co.-.l)elilion of 12sI-AIV binding to AT4 receplo.
in CVEC membrane prel~a ~lions Fragment Sequence Kj AIV VYIHPV 1.1 +/- 0.2nM
AII(~-7) VYIHP 7.3 +/- 1.2nM
Am RVYIHPF 23.3 +/- 3.4nM
AII(, X) DRVYIHPF 193.8 +/- 44.5nM
AII(4-~) YIHPF 252.6 +/- 89. lnM
Sarl,Ile~-AII SRVYIHPI 261.0 +/- 89.1nM
DUP 753 --- >10~
CGP 42112A --- >10 4 *AII values lc~resenl mean +/- SEM of two e~el..ll~llLs with duplicate samples; K
determined by LIGAND.
G-protein linka~e of the AT4 receptor in vascular cells G-protein interactions with vascular angiotensin receplo,~ are shown in Figure 9, where ".~;...I,.ane fractions from rat vascular smooth muscle cells (RVSMC) or bovine aortic endothelial cells (BAEC) were preinc~lb~ted in various conce~ aLions of a non-hydrolyzable GTP analogue (i.e., GTP~S) for 60 mimltes at 22C prior to WO 94/00492 PCr/US93/0603 21391~ -62-use in equilibrium binding assays with 0.5nM 125I-AII (RVSMC) or 0.6nM l25I-AIV
(BAEC). (No.l of exper. =3; each with duplicate samples.) Data presented here represent results from a single experiment.
Addition of non-hydrolyzable GTP (i.e., GTP~S) to the binding assays did not 5 inhibit (or alter) binding of l25I-AIV to AT4 receptors in BAEC lllelllbl ~1e prep~aLions (Figure 9). In constrast, in a positive control GTP~S inhibited 125I-AII
binding to AT1 receptors in rat vascular smooth muscle cell (RVSMC) lllelllbl~ eple~ ions in a dose-dependent manner (Figure 9); in a~,lt;~lllenl with observations reported previously by others. (This property flietin~ hes AT4 receptors of the 10 invention from ATl and AT2 receptors reported by others previously in vascular tissues.) Discussion This study is the first to describe a novel angiotensin binding site in vascularendothelium that exhibits high affinity and specificity for the h~ apeplide AIV
15 fragment of angiotensin AII. The AT4 receptor is distinct from the AT1 or AT2receptors in vascular tissue. Analysis of the binding characteristics intli~ctes that the AT4 receptor binds AIV in a saturable and reversible manner, and that l25I-AIV
reaches equilibrium in binding to the AT4 receptor in lllellll,.~e plep~lions inapploAilllalely 60 min. at 37C. Binding of AIV to its receptor lelllaills stable for at 20 least 4 h (Figure 7A) ~,vith less than 10% degradation of the ligand under these binding conditions. Scatchard analysis of the AT4 receptor binding site by the non-linear curve fitting program LIGAND reveals two collll)onellls to the binding data. The first colllpol1elll is a high affinity component that e-Ahibits Kds of 14 and 27pM with BmaX~s of 6 and lOfmol/mg protein for receptors in CVEC and BAEC Illt;lllbl~-e 25 prep~lions, le~ecLi~ely. (Because of the t;A~l~lllely low number of these high affinity sites it is unclear at present whether this is a physiologically important state of the receptor; or, is a result of modification of AT4 rec~ol~ in the nle~ ne prep~lions, or ch~npes in receptor binding affinity reslllting from co-operativebinding of AIV; or alternatively, that this site is an artifact created in the melll~ e 30 prep~alions or assay con~ition~-) The second binding collli)ontilll is a lower affinity cGlllpol1elll with Kds of 1.4 and 4.4nM (i.e., in CVEC and BAEC, resl,e~ ely). The second corlll)on~.ll d;s~ a high concellllalion of ligand binding co, .""e~ lrate with large numbers of such receptor sites in the Ill~ e prep~lions: i.e., these sitesbind 594 and 434fmol/mg protein in CVEC and BAEC lllenlbl~-e plel)a,aLions, 35 respectively.

~0 94/00492 2 1 ~ 9 1 0 5 PCI/US93/06038 -63~

The overall binding affinity (i.e., Kd, single or composite site fit produced byLIGAND) was calculated to be 0.7nM for CVEC and 1.0nM for BAEC. These results are in good agleelllt;llL with the Kd calculated from the results of kinetic binding studies (0.3nM).
The pharmacological profile derived from co~ ;LiLion displ~cem~nt of 125I-ATV bound to these AT4 receptors in vascular tissues is presented in Figure 8 and Table 11, above. This profile reveals a strict structural requilt;lllelll for the N-terminus of the AIV ligand, i.e., removal of the N-terminus (Vall) of the AIV
ligand results in a 200-fold decrease in affinity of the AIV ligand for the AT4 receptor in vascular tissues (i.e., an increase in the Ki). In addition, N-terminal extension, i.e., beyond Vall, is d~llh,~ l to the binding of AIV ligands to the vascular AT4 l~cel,lor as inr1ir~ted by the inability of AII and Sarl,Ile8-AII to co---l.eLiLi~ely inhibit binding of AIV to the AT4 t;cel.lor, (i.e., note the 200-fold increase in Ki seen with AII and Sarl,Ile8-AII, when co..l~aled with AIV in Table 11). (This property 15 ~iictin~liches AT4 receplGls of the invention from AT1 and AT2 receptors.) The app~c;.-l affinity of Am for the vascular AT4 receptor (i.e., 20-fold higher Ki than AIV, Table 11) may be an artifact of N-terminal metabolism of AIII to form AIV in these --t;---b-~le prep&,~lions. (In previous studies, above, 125I-AIII binding to bovine adrenal AT4 receptors was directly plopo-lional to the amount of Am 20 hydrolyzed to AIV.) The vascular AT4 receplor appears to exhibit less sper.ifi~.ity for the C-terminus than exhibited for the N-terminus: i.e., the AIV(1 7~ fragment (with the C-terminal Phe8 deleted still bound with re~oll~ble affinity to the lt;ce~llor (i.e., only a

7-fold increase in Ki over AIV). (These r~ ii"gs are in ag-ee...t..l with the fintling~
25 above in Example 1 using AT4 receptors in bovine adrenal cortical tissues.) The vascular AT4 r~ce~lo.~ do not apparelllly bind either DUP753 or CGP 42112A (i.e., Ki >10 ~), but ATl or AT2 receptors are well-known to do so (Ti.. t.. ~1s, P. et al. 17P5 12:55-62, 1991; Whilel~.~zd, S. et al. Biochem. Biophys.
Res. Comm. 163:284-291, 1989). (This propc;.ly offailure to bind either DUP 753 or CGP42112A .li~tin~ hes AT4 rece~ol~ of the invention from AT1 and AT2 receptors.) Bil-di-1g of 125I-AIV to vasular endothelial AT4 rect;plo-~ was not sensitive toinhibition by f~l~nine nucleotides. In contrast, binding of AII to AT1 and AT2 receptors in .n~;.nb.~le prep~lions of rat vascular smooth muscle cells (RVSMC, Figure 9) was sensitive to inhibition by guanine nucleotides in a dose-dependentmanner, i.e., the affinity of the AT1 receptor for AII was shifted to a lower value Wo 94/00492 PCr/US93/06038 2~3g~0S
when the receptor was uncoupled from G-proteins by the presence of the GTP
analogue GTPyS (Figure 9). This shift in binding affinity in response to gunainenucleotides is a characteristic of the high affinity form of the AT1 receptor (Gloccm~nn H. et al. J. Biol. Chem. 249:664-666, 1974). The in~n~itivity of the 5 AT4 receptor to G-protein uncoupling agents was also observed with AT4 receptors in membrane p,t;pal~lions of bovine adrenal cortex. (This plopel ly of imçn~ifivity to G-protein uncoupling agents (lictin~ hes AT4 lecep~ol~ of the invention from AT1and AT2 receptors.) Despite the inability of AIV to bind to AII receptors, several recent studies 10 have sllg~ested that that AIV-like fr~m~nt~ of AII may have unique biologicalattributes. In cultured chick myocytes, AIV-like fr~ ntc of AII have been reported to antagonize the effects of AII-indllced increases in cytosolic free c~l~.illm protein synthesis, and hy~elLlopl~ic cell growth while being unable to cc,lllp~liLi~ely inhibit for 125I-AII binding (Baker, K.M. et al. Am. J. Physiol. 259:H610-H618, 1990). Topical 15 application of both AII and AIV-like fra~m~nte of AII have been reported to me~i~te endothelium-dependent vasodilation in rabbit brain arterioles. However, in the presence of the amino peptidase inhibitor ~m~ct~tin the vascular response to AII, but not AIVlike fr~gmPntc, was reportedly blocked (Haberl, R.L. et al. Circ. Res.
68:1621-1627, 1991). AIV-like AII fr~gmPntc and AII have also been reportedly 20 applied intracerel)lo~entricularly in the rat where they reportedly are equipotent in enh~nt~.ing memory and learning (Braszko et al. Brain Res. 542:49-54, 1991). Given the low affinity of AIV for ATl and AT2, disclosed herein, it is most likely that the latter activities previously attributed to binding of AII and/or AIV-like fr~gm~ntc at AT1 and AT2 sites are, in fact, the result of binding of AIV at the AT4 receptor sites 25 of the invention.
It is likely that the actions evoked by AIV binding to its specific AIV recepotrs may act collll~y to the actions ofthe AII and AT1 and AT2 receptors. For example, infusion of AIV into rat kidney, as shown above, to stimlll~te a significant increase in blood flow in the renal cortex, while AII binding to AT1 and AT2 recep~ol~ in these 30 tissues produces the converse effect - a cignific~nt decrease in blood flow.
Effects on Vascular Tissues:
~ seC~ of AIV effects on the contractile plopelLies of aorta and inferior vena cava was delllol1s~la~ed using tissues from rabbits. The presence of numerous AT4 receptors in aortic tissue suggest a possible action of AIV ligand on cerebral 35 vessels. The routine use of rabbit aortic strips or rings in cardiovascular pharmacology dictate that rabbits are suitable for use in such studies.

~VO 94/00492 2 1 3 9 1 0 5 PCI/US93/06038 The following protocols are useful for: 1) co"r",l.ing the vasodilating potential of an AIV ligand, delllo~ laLi,lg that ligand action is dependent on an AT4 receptor, and showing that the action is independent of AI or AII receptors;
2)establishing that any observed vasodilation is endothelium dependent;
5 3) dete",~ g whether the me~.h~ ", of vasodilation involves prost~gl~n~in.c, EDRF, or other factors like EDHF as second ...ess~l-g~.~; and 4) determining thefunctionality of the many AIV analogues (i.e., such as those synthesi7ed in Example 4) as either AIV ligands or as agonists, antagonists, inhibitors, or promoters of the AIV ligand-receptor interaction.
AIV and AII ligands and various analogues (Example 4) in the presence or absence of angiotensin inhibitors (e.g., Sarl,Ilex-AlI, DUP 753, and CGP42112A) were screened for the vasodilating activity using rabbit aorta and inferior vena cava rings or spiral strips suspended in 20ml organ baths co..~ ,;..g Krebs solution at 37C
and continuously gassed with 5% C02 in oxygen. After a 1 h equilibration period,c.-mul~tive dose-re~onse curves were constructed for the analogues over a concentration range of 10-10M to 10-sM. In relaxation studies, aortic strips were pre-contracted to 70% of m~im~-m di~met~r with phenylephrine, and then the test ligand is added and relaxation of the vessel is qu~ntifiçd Changes in contractile or relaxant response may be c~lcul~ted for each dose of each di~erell~ ligand or analogue and subsequently analyzed by analysis of variance.
Effects on Endothelial Cells:
The effect of AIV ligand on endothelial cells was ~ .";.~ed by measuring growth of bovine endothelial cells. Cells were grown at 37C in 35mm culture plates C02/air under 5% C02/95% air in Dulbecco's Modified Eagle's Medium (DMEM) suppl~mented with 511g/ml insulin and 10% (v/v) newbornbovine serum (NBBS).
The test metlil-m was suppl~m~nted with 3H-thymidine and either AII ligand (50nM) or AIV ligand (SOnM) or lOng/ml acidic or basic FGF (as a positive control).
Negative controls were also inc1uded using ethi~1i--m bromide (lmM). The cells were harvested at various times, and cellular lysates were prepaled for sçintill~tion counting by lysing and waslllllg the cells on glass fiber filters.
Materials and Methods:
ntS
AIV (VYIHPF), AII(3 7~ (VYIHP), and AII(4 8~ (YIHPF) were synth~si7ed as described in Example 1, above. All reagents and other peptides were obtained from Sigma Chemical Co., with the exception of: Plummer's inihibitor (Calbiochem);
bestatin (P~ninc..l~ Biochem); DUP 753 was a gift from Dr. Ron Smith of WO 94/00492 -1 0 ~ -66- PCI/US93/06038 Dupont/Merck and CGP42112A was a gift from Dr.Marc deGasparo of Ciba-Geigy. Angiotensin fragmPnt~ numbering was based on the sequence of AII
(Figure 1).
Cell Culture Bovine coronary venular endothelial cells (CVEC) were isolated by a bead-perfusion technique and characterized as described previously (SçhPllinE M.E. et al.
Am. J. Physiol. 254:H1211-H1217, 1988). Bovine aortic endothelial cells (BAEC) were a gift from Dr. Stephen Schwartz (University of Wa~hinEton). Cells were grown in 100mm tissue culture plates (Falcon, Becton Dickinson Co.) coated with 1.5% gelatin in PBS (per liter of distilled water: 8.12g NaCL, 1.14g Na2HPO4, 0.28g NaH2P04) in Dulbecco's modified Eagle's mç~ lm (DMEM; FLOW Labs) s.lppl~ ed with 2mM sodium pyruvate, 2mM L-El-~t~mine., 100mglml heparin, 100mg/ml Penicillin-G, 50mg/ml SlreplolllyGin, 44mM NaHC03, and 10% fetal bovine serum (GIBCO). Cells were passaged 1 :3 by tryptic digestion (0.05% trypsin, 0.025% EDTA in Ca++/Mg~-free PBS, pH7.4 at 37C). All data collected in this study was from cell lines p~s~Eed between passage 5 and passage 9.
Tissue pl t;?~u ~lion Cells were grown to confl~çnce in 100mm culture dishes. Dishes were washed once in Ca++/Mg~-free PBS, pH7.4 at 37C follwed by the addition of 2ml of cold isotonic assay buffer (150mM NaCI, 50mM Tris, lmM PMSF, 1011M bestatin, 50~M Plummer's inhibitor, pH7.4 at 4C). Cells were then removed from the plateswith a rubber policeman and homogenized in 5ml assay buffer for applo~illlalely 10 sec (Polytron, Brinkman Inst. Co.). Cell extracts were centrifuged at 40,000 x g for 20 min at 4C, the ~upelllal~ll was discarded and the pellet was rehomogenized in assay buffer and centrifugation was repealed for a total of two high speed centrifugation steps. The final pellet was resuspended in assay buffer to a working col~ce~ a~ion of appr~.,.;...~P.Iy Smg/ml as delelll~ed by the method of Lowry (J.
Biol. Chem. 193:265-267, 1951).
Iodination of AIV
AIV (and other peptides) were io-lin~ted using an immobilized lactoperoxidase-glucose oxidase system (Enzymobeads, Biorad Laboratories) to a specific activity of 2176Ci/mmole. l25I-AIV was separated from unlabeled peptide by HPLC (Beç~ n) using a reverse phase Cl8 column (5mm x 250mm; Adsorbosphere, Alltech, Associates).

Receptor binding assays Binding assays were performed at 37C in a total volume of 250ml (isotonic buffer, pH7.4 at 37C). Bound and free ligand were separated at the conclusion of each eAI~e~il.lent by the addition of ice-cold PBS (pH7.4), and separation of bound 5 from free was achieved by 4 vacuum filtration washes with 4ml of this buffer (Schleicher and Schuell #32, Brandel Cell Harvester). Radioactivity retained by the filters was determined using a Tracor Analytic gamma counter, model #1185 having68% counting efficiency. Nonsl,e~iific binding was ascellailled in the presence of lmM unlabeled AIV.
Kinetic binding ~iAI~elilll~llLs (N=3) were pe,f~""ed at 37C over a time courseof 240 min with 11 time points and duplicate s~ p!es. The appale..l pseudo-firstorder association rate consl~.~ kob5 was detc.;..,ined by the non-linear curve fitting program LIGAND. Dissociation eA~e i..-e.lLs (N=4) were conrhlcted at 37C by pr~incub~ting cell eAtracts for 120 min with 0.5nM radiolabeled ligand followed by 15 the addition of lmM unlabeled ligand (final conc.). Binding was de~e-....lled for duplicate samples represe..l;ng 10 time points over 180 min. The appale..L
dissociation rate consl~-L, k l, was dett-.. ined by LIGAND. The appare..L
association rate consL~.L, kl, was then c~lcul~ted from the equation k1= (kobs - k 1)/[L], where [L] is the radioligand concellLlaLion~ and the apparenL kinetic 20 equilibrium dissociation consL~u.L~ Kd, was derived from the equation Kd = k l/kl.
Saturation equilibrium binding and col..peLiLion diepl~cemrnt studies (with CVEC, N=4 expts., 46 total data points; BAEC, N=3, 34 data points) were conrlucted over 120 min. of inr,ub~tion in the presence of incl~,asing conce--L-~lions of radioligand or co...~,elh~g ligands, ~e~;Li~ely. Saturation data were analyzed by LIGAND for the dt;Le.. linaLions of .. ~ ;.. number of binding sites (Bma,~) and Kd.
For delt",--l~,g the linkage of G-proteins to the AT4 receptor, l~l~lllblane - prepa~aLions were first pr~inruh~ted in GTP assay buffer (50mM Tris, 150mM NaCI, 5mM MgCi2 lmM EGTA, lmM PMSF, SO~M Plun~ner's inhibitor, lO~M bestatin, p~I7.4) at 22C for 60 min in solutions of GTPyS ç~ ted to produce a final concenLlaLion in the assay of lOOmM, lOmM, lOnM and 0 GTPrS. The rat vascular smooth muscle cell line WKY IV passage #17, was inr~ 1ed as a positive control for G-protein linkage to ATl lecel)lo.~. All data are plesenled as the mean +/- SEM,standard error of the mean.
Endothelial cell growth and the effects of AIV ligand on EDRF production Bovine aortic endothelial cells were grown at 37C in 35mm culture plates under 5% CO2 in air in Dulbecco's Modified Eagle's Medium (DMEM) supplrmented WO 94/00492 2~39~Q PCI/US93/06038 with 5~1g/ml insulin and 10% (v/v) newborn bovine serum (NBBS). The m~ m was aspirated 10-12 hours after seeding and replaced with serum-free medillm The me.1illm was again aspirated 10-12 hours later and replaced with either test or control metlillm Control medillm was DMEM with 5g/ml insulin and 2%, 5%, or 10% (v/v) NBBS as indicated. The test medillm was supplçm~nted with either AII or AIV
ligand ~ the antagonist Sarl,Ile8-AII at various concentrations. The merlillm was çhAI~ged every 48 h (i.e., with supportive DMEM metlillm for the r~mAinrler of the ~e-illlent).
For measurements to determine the effects of AIV in stiml~lA~ting an increase inendothelial cell numbers, cells can be harvested on various days during the culture period by washing the plates with calcium free mç-lillm (CMF) two times for 5 min.
followed by in-,ub~tion in 0.1% trypsin in CMF for 5 min. The cells can then be washed free from the plate and as~ ed by Pasteur pipet into 15ml centrifuge tubes cG,~I~;n;,~g 3ml DMEM with 20% (v/v) NBBS. The plates can be washed with an additional lml DMEM 20% NBBS which was Ll~lsr~lled to the appropl;ate centrifuge tube and spun at 300 x g for 10 min. Excess metlillm was aspirated and the pellet resuspended in a final volume of lml of the control metlillm Aliquots can then be counted using a hemocytometer and cell number eA~,lessed as cells/plate.
As an adjunct to the detellllllld~ion of cell llulllbel~, thymidine incorporation was measured. For qu~ntit~tion of DNA synthesis [methyl-3Hlthymidine (60Ci/mmol,1 OmCi per plate) was added to cultures 12 h after addition of the AII or AIV. Twelve h later, medillm was removed and lml of a 1% aqueous solution of Triton X-100 was added. The cells were inr,~lb~ted with this solution for 5 min. and the entire contents of the plate Ll~l~r~ d to lOml of absolute ethanol. This material was then filtered under vacuum through 2.4cm glass fiber filters (GF/A~ Whatman), and the filters were washed twice with lOml of absolute ethanol and assayed for radioactivity by sçintill~tion counting.

Physiological Function of Angiotensin IV Receptor and Ligand Angio~ensins AI, AII, and Am are reported to have a wide variety of effects on target issues, some of which are acute while others appear more long-term. AII
reportedly has a cellular effect of incleasing c-fos levels in cultured vascular smooth muscle cells (17), and c-fos is reported to be one common pathway for triggering cell growth. Considering the widespread distribution of AT4 receptors in many organs and tissues (EXAMPLES 1 and 2, above), it is likely that AIV has multiple functions, ~0 94/00492 ~ . PCr/US93/06038 inrl~1ing long-term effects on cells by triggering increased ~A~uression of c-fos, i.e., activities previously miet~k~nly attributed to AII and AIII.
The following studies focus on the role that the AIV ligand-receptor system may play in three organs enriched in AT4 rece,ulola: blood vessels, kidney~ and adrenal glands. (Other organs such as brain or heart which also possess high levels of specifically localized AT4 receptors can be studied in a similar manner.) Renal Blood Flow: The AIV Receptor and AII Receptor Have Physiolo~ically Distinct and Opposin~ Activities:
Physiological studies, des~il;bed below, investig~ted the involvement of AIV
ligand in the regulation of renal blood flow. The rationale for initially choosing to f~x~mine the kidney was at least two-fold. First, the AT4 receplor is found in high conce~ ions in kidney and endothelial cells (Example 1 and 2, above). Second, vascular endothelial cells are reported to regulate vascular tone and to play a role in the control of renal blood flow.
Superficial blood flow in the rat kidney was ~sç~ed using laser doppler methods in ~nestheti7ed rats following direct infusion of a test substance into the renal artery. The results are presented in Figure 4 which depicts the pel ce--lage change in cortical renal blood flow following infusion into the renal artery of 25~1Vmin of a 0.15M NaCI solution co..l~ g 100pmoV25111 AIV (closed circles; number of eA~e in-e-"s (n)=13); 0.15M saline (open circles; n=9); 100pmoV25111 of AIV lacking the N-terminal Vall residue (i.e., YIHPF; D-Vall; closed squares; n=9); and 100pmoV25~1 of AII (open squares; n=8). The infusion of eAI elillltillLal compounds and saline had no effect on systemic arterial blood pressure (see results in Example 4).
The infusion of AIV (closed circles) show that AIV ligand infused at 100pmoVmin.25 stiml~tes a ploround and long-lasting increase in blood flow. In contrast, infusion of AII (also at 100pmoVmin.; open squares Figure 4) produced a dramatic decrease inrenal blood flow. The AIV analogue d-Vall-AIV (i.e., lacking the N-terminal valine and lacking binding activity for the AT4 receptor, see F.y~mple 1, above) had noeffect on renal blood flow (closed squares; Figure 4).
The eA~ enlal protocols employed in these studies is det~iled in the Materials and Methods, below.
Materials and Methods:
EAIJel;ll1elllal Protocol #1:
For co---p~;son of AII, AIV, d-Vall AIV and saline infusion on renal blood flow, the rta~ue~ re agents were infused into the renal artery at 100pmoVmin. for 10 min. at 25~Vmin. Saline and the AIV analogue d-Vall AIV were inr.l~lded as WO 94/00492 ~39~oS PCr/US93/06038 controls, i.e., the number of cA~ illlel~ 8; average standard error of the mean (SE) = + 3% change blood in flow. As expected, saline and d-Vall AIV had no effect on renal blood flow. Also, as expected, AII produced a dramatic decrease in flow followed by an autoregulatory return toward baseline. AIV produced an equally 5 dramatic increase in flow that showed little autoregulation.
Consistent with the involvement of di~renl receptors in the mediation of AII
and AIV effects, the specific AII antagonist Sarl,Ile8-AII (lnmol/min - lOmin.
p~Lle~,.,P.nt) completely blocked the AII effect while having no effect on AIV. The decrease in blood flow w;~ ed with AIV was dose dependent and was not 10 accG..~p~Ilied by alterations in mean arterial pres~ure, sllg,~e~ g that the effects of the AIV ligand-receptor system may be limited to selective vascular beds or that co~ .s~lory ~.h~ng~ in cardiac output occurred during AIV infusion.
EA~;,i".e."al Protocol #2:
The AIV-in-luced increase in renal blood flow was not blocked pr~infi.~ing 15 AII: Sarl,Ile8-AlI was infused over the 10 min. immedi~t~ly prior to infusion at lnmol/min., and a COIllp~iSOll was made with the change in blood flow that occurred when AIV ligand was infused will~oul the AII preinfusion. In 8 expe~i",~"~s an average change in AIV-in~uced blood flow of <3% was recorded with the AII
preinfusion, which was within the ~L~ da,d error of the cA~,i",ellls, i.e., SE = _ 3%.
20 Thus, as predicted from the co""~liLion binding studies con~1cted above (Example 1), Sarl,Ile8-AlI was unable to block the vasodilatory effect of AIV ligand.
When tested in control cA~t;,i",ents for the ability of AIV ligand to block AII-medi~ted decrease in blood flow (i.e., in the same type of preinfusion eAI,e~i",e,lL, but using AIV preinfil~ion instead of AII). AIV ligand completely blocked the 25 constrictive action of AII. The,eru,c;, the results support the notion that AIV may antagonize certain of the actions of AII.
Effects of AIV-Ligand-Receptor Interactions on Renal Functions Results presented above dç~..ol.~l.a~e that intravenous application of AIV
ligand can dr~m~tic~lly inclease renal blood flow and urine flow in a dose-dependent 30 fashion. This effect appears to be metli~ted by the AT4 receptor and not by nollspeciflc, nol~rece~"or-dependent processes. Neither AII nor d-Vall AIV (a nonbillding AIV analogue) could reproduce the effects of AIV ligand, and the specific AII antagonist Sarl,Ile8-AlI was unable to block the action of AIV ligand.
Another ~çc~ ..l of the AIV ligand-receptor effects on renal functions was 35 provided by analyzing distribution of radio-labeled insulin and p-~minohirpicuric acid;
in coll,bh-a~ion with ,lleasw~",e"~s of urine flow, urine osmolality, urine Na+ and K+, ~vo 94/00492 2 1 3 9 1 0 5 PCI/US93/06038 --71- - ~.

- and hematocrit. The effects of AIV ligand, AII, and other AIV analogues were determined, i.e., a) on renal blood flow, b) glomerular filtration rate, c) osmolal clearance, d) filtration fraction, and e) tubular function. Dose-response curves for AIV ligand and AII ligand were constructed in the presence and absence of the AII
5 antagonist Sarl,Ile8-AII. In addition, AIV analogues with special in vitro properties (e.g., AIV antagonists, AIV superagonists, or metabolically IGs;sL~lL analogues of AIV) were tested in a similar manner (above) to determine their effects on renalfunction. Studies were carried out as acute prepal~Lions in ~nestheti7ed rabbits and using jugular and urethral c~thetçrs.

Neurological Effects of the AIV-AIV Ligand-Receptor Interaction Local Effects:
Given the plGsellce of AT4 receptors in the brain (Example2, above;
Figures 6-10) and most likely in cognitive and motor memory and learning centers(i.e., hippocampus, frontal cortex, cerebellum, and th~l~ml-s), and in areas within the hindbrain cardiovascular nuclei involving the tractus solitarious, it is reasonable to suspect that at least in some tissues AIV ligand is produced locally in neural tissues, i.e., by synthesis of Al and conversion to AIV. Two scenarios of local production can be envisioned. In the first, AIV ligand is produced locally from precursors synthP~si7ed in the tissue. In the second, circ~ ting AIV precursors (e.g., Al, AII or Am) are converted locally to AIV ligand. Whether the first or second scenario is an operative ...e~.h~ni.cm in a particular tissue can be d~LG.I..~lled by introducing radiolabeled precursors (i.e., l25I-AI) into the bodily fluid bathing the tissue (e.g., plasma or CNS fluid), and by then collecting samples of the fluid at di~erG..L times and 25 assayil~g by reverse-phase HPLC to d~Le~ e if the AIV precursor has been converted to AIV ligand in the fluid. If it has been converted, the second scenario is operative; if it has not been converted a second series of ~ e-h.ltllLs is cond~lcted In the second series of e.~GlilllGnls bios-ynthesis of AIV precursors is evaluated (i.e., with radiolabeled amino acids) and conversion of the precursor into AIV ligand is 30 e~ ed in pulse-chase type ~ G,hllellLs. If biosynthp~ti~lly radiolabeled AIV
precursor chases into AIV ligand, then the first scenario is operative in the tissue.
Changes in the AIV-Ligand-Receptor System in Response to Neurological Effects:
A represGllLaLi~te e,~l~tl;lllGIlLal protocols for showing çl~ngP~S in the AIV-ligand-receptor system in re~uonse to neurological and physiological effects is 35 described in the Materials and Methods, below.

W O 94/00492 PC~r/US93/06038 ? 13 g 1~5 -72-AT4 receptors in brain:
A co"l?~ison was made of the binding affinities (under equilibrium binding conditions) of AT4 receptors in dirrelell~ regions of guinea pig brain. The results of Scatchard analysis of binding data (con~ucted in the manner described above in 5 Example 1) are sullllllali~ed in Table 12, below.

Binding of AIV in Regions of Bra na Brain Region Kf~ (nM) Bm~X (finol/mg) HSTAb 0.11 +/- 0.051 168 +/- 52.7 Hippocampus 0.10 +/- 0.073 306 +/- 95.1 Cerebellum 0.21 +/- 0.237 232 +/- 93.2 Brain stem 0.09 +/- 0.054 197 +/- 63.9 a.) mean +/- SD; no. of c,.~ ls =4 b.) HSTA= hypoth~l~mlls, th~l~mllc, septum, antereoventral third ventricular area.
10 The Hippocampal AIV Ligand-Receptor System:
Hippocampal AT4 receptors identified in tissues by receptor autoradiography in Example 2, above, were evaluated further by isolating hippocampal Illt;lll~ es (i.e., inr.l~ltling hypoth~l~m..s, th~l~m..c, septum, anteroventral third ventricular area, HSTA, above) and then solubilizing the receptor. (A similar approach may be 15 employed with AT4 receptors in other tissues.) The results presented below show that the guinea pig hippocampal AT4 receptor binds AIV ligand with a high affinity (Kd= 1.29+0.18nM, mean +SD, Hill Coeff. = 0.993+0.015) and in a saturable manner (Bmax=449+62fmol/mg protein). (It is not~wolllly that the guinea pig hippocampal AT4 receptor binds AIV ligand with applo~ill-a~ely the sarne binding20 affinity as the bovine adrenal AT4 receptor desc,il,ed in F.Y~mple 1, above.) The density of the AT4 receptors in hippocampàl cells and tissues was considerably higher than l~l,olled in brain for AII receptors(43,44). In the present studies no AII
receptors could be detected in Hippocampus by binding of 12sI-Sarl,Ile8-AII (data not shown). The N-terminal structure of the binding AIV ligand is pal~l,olmt in 25 dt;~t;l,l~ling the binding affinity. The C-terminal requilel~lenls seem less ~lh~gt;ll~ as evidPnr,ed by the binding affinity of AII(3 7~ (Kd = 20.9 + 2. lnM). Neither AII, AIII, Sarl,Ile8-AII, Dup 753 nor CGP42112A appear to bind intlic~tinp that this binding site is neither the ATl nor AT2 sites described for AII/AIII. Autoradiographic analysis of Hippocampus binding COI~lllS the inability of Sarl,Ile8-AII to 30 colllpt;~ ely inhibit for l25I-AIV binding. Conversely AIV was unable to displace `~0 94/00492 2 1 3 9 1 0 5 PCI/US93/06038 -7~ -l25I-Sar1,Ile8-AII binding at this site. The finding of AT4 receptors in the Hippocampus s~1ggestc that AIV ligand-receptor interactions may merli~te unique central angiotensin-dependent functions inr~ ling memory rnh~nr~em~nt and provide a link between the Hippocampus and memory.
Saturation isotherms and corresponding Rosenthal plot for l25I-AIV binding to AT4 receptors in guinea pig hippocampal ~ lllbl~nes show specific binding of 125I-AIV ligand to isolated hippocampal lllt;lllblane AT4 receptors purified from guinea pig brain. Nonsl)ecirlc binding was defined in the presence of non-labeled competitor, i.e., lOOnM AIV. The eA~ lenl was carried out 5 times (n=5);
125I-AIV bound saturably and ligand analysis of the binding data indicated the presence of a single high af~inity binding site (Kd = 1.29 + 0.18nM), Bll~aX = 449 ~ 62 femtomol/mg protein; Hill Coef= .993 + .015; mean + SD.
Structural characteristics of AIV ligands that d~elll~ine binding to the hippocampal AT4 receptor were det~...;.ed in colll~,~Lilion binding studies, i.e., 15 similar to those described above in Example 1. The results of these competition studies are presented in Table 13.

Colllpe~ilion of 125I-AIV Binding -o Guinea Pig Hippocampus Melllbl~les*

Compound Kj (M) AII <10~
Am 1.60 + .09 x 10-7 AIV 4.28 + .51 x 10-9 AII(~-7) 2.09 + .45 x 10-8 AII(4 ~ >10 AII(~ ~ >10 Sarl,Ile8-AII >10 Dup 753 >10-4-CGP42112A >10-4-*n = 2, mean_ SD; 25mg of total lll~e protein was inr,~lb~ted with 0.6nM
20 125I-AIV plus a variable concellLI~lion of unlabeled angiotensin as a colllpc;~ilor The results of these studies confirm those pre3ellled above in Example 1 with bovine adrenal AT4 rece~lol~. The N-terminal of the AIV ligand (e.g., valine) is a major detellllil~ of binding affinity. In agreement with the saturation isotherm data, AIV
exhibited a high specificity for AIV (Table 13). N-terminal extended peptides 25 inrl~ltling Sarl, Ile8-AII, AII, and Am had significantly reduced affinities for the AT4 WO 94/00492 2~39~QS PCI/US93/0603 receptor while AII(4 8), which has the N-terminal L-Val removed, did not bind. (The low, but apparenl ability of AIII to bind, may (as above) be due to conversion of AIII
to AIV. The C-terminal specificity of the hippocampal AT4 receptor appears less.Removal of Phe from the C-terminal of AIV ligand diminieh~e, but does not ~ e 5 binding (Table 13), while removal of Phe, Pro, and Ile ~limin~tes binding. As seen in Table7 neither Dup753 nor CGP42112A colllp~ ely inhibited for the binding of 125I-AIV to the AT4 receptor. In addition, the peptides listed in Table 14, failed to bind to the HIV rece~lor in guinea pig brain as evidenced by their inability to significantly alter binding of AIV to receptors in this tissue.

Nonbinding Peptides (K,l >lO~M)a ~pGlu,Cyt,~l-AVP(4 9) pmp, O-Me-Tyr2-Argx Argx -Vasopl essi Ne:urolellsin Oxytocin Substances P
VIP
Neuropeptide Y
Atriopeptin TRH
Tetradecape~Lide Met-Enk Leu-Enk Gly-Phe-Ala Bradykinin a.) Peptides that fail to bind to guinea pig brain tissues as evidenced by Kd >10~M.
This study demonstrates the exiet~once of a unique angiotensin binding site in guinea pig Hippocampus which is specific for the N-terminal deleted AII hc~eptide, 15 AIV. The location of this specific binding site in the Hippocampus supports the hyl~olllesis that the AT4 leceplor is the receptor that metli~t~s angiotensin-dependent cognitive effects in the brain. It is clear from the autoradiographic sections shown in Figures 6-10, above, that the l25I-AT4 receptor is not restricted to the Hippoc~mpus.
The localization of 125I-AIV binding sites in other brain regions det~iled in Table 15 20 presents an OppOI lullily to expand the realm of angiotensin AIV-related actions.

``'O 94/00492 2 1 3 9 1 0 S PCr/US93/06038 Autoradiographic Qu~ntit~tion of AIV Receptors in Brain AIV Displaced by AII
Regiona AIV Bound (fmol/gm) (fmol/gm)b Cerebellum 6950.7 +/- 1675.7 6122.2 +/- 1496.3 Hippoc~mpus 5059 +/- 1963.7 4501.8 +/- 223.0 P;lir~llll Cortex2771 +/- 954.7 2605.6 +/- 789.2 Par 1/2 1644.3 +/- 343.5 1710.9 +/- 369.8 Fr 1/2 1551.9 +/- 604.2 1446.2 +/- 453.6 Ca~ te Putamen1755.1 +/- 622.1 1663.3 +/- 654.1 HDB 2082.3 +/- 702.2 1985.6 +/- 621.1 Th~l~ml.s 2077.9 +/- 390.9 1904.4 +/- 646.5 Inferior Colliculus 2432.1 +/- 871.49 2235.0 +/- 663.8 SOL** 2446.3 +/- 881 2053.6 +/- 714.9 ION** 3323.1 +/- 136.3 3267.7 +/- 461.0 a.) n=4 t:AI~Clilll~ S; *n=3 t~pelhll~llls; **n=2 e"~.efil~ s, b.) displ~cem~nt of 125I-AIV by Sar,Ile-AII
5 Cognitive effects of the AIV ligand-receptor system Lea~ g. The results presented in Figure 10A show the mean latency (sec +/- SEM) for indepelldellL groups of rats to re-enter the dark colll~ lllell~ on Days 2-4 following passive avoidance conditioning on Day 1. One minute prior to the shock trial on Day 1, lllelllbel~ of each group received aCSF (2ml), or 100pmol in a 10 total volume of 2ml aCSF of AII or AIV. On subsequent test days each animal was placed back into the lighted CGlllp~h llllelll and latency to enter the dark COlll~ lllent was measured. Members of the group that received AIV on Day 1 showed .~iEnific~ntly elevated latency times to re-enter the dark side on Day 2, as colll~ ed with the mean results from animals in the aCSF and AII test groups. On day 1 15 artificial cel ~rospinal fluid (aCSF), AII, or AIV was ~11mini~tered by intracel~broventribular (icv) injection into rat brains one minute prior to training.
Training was con~litiQned (as desribed above) to avoid a dark CCilll~ llllenl. On Days 2,3, and 4 of the c,~l~c;lilllc;lll the animals were tested for the latency of time before they would re-enter the dark colllp~lmelll. F.nll~nc~ of memory retrieval was 20 observed on days 2 and 3 after learning of the reponse (Figure 10B). As can be seen WO 94/00492 ` PCr/US93/0603 213910~ -76-from the results presented in Figure 10A the effect diminiched with time after the learning of the response.
Memory Retrieval: The effects of AIV ligand on learning and memory were tested in rats by measuring the passive avoidance response, i.e., the mean latency period (time in seconds) for which the animal avoided a dark COlllp~ l",ent. Training was conditioned to avoid the dark Colllp~Llllt;ll~ by a(lminictrring a 0.25mA foot shock over a period of 2 seconds with the door to a lighted COIlllJal Lll1~11L closed. On day 2 retrieval of the cognitive memory was tested 5 minllteS after intracel~l~ entribular (icv) injection of AII or AIV. The results presented in Figure 12A show that AIV has a positive effect on memory retrieval at lnmol and 100pmol, i.e., the AIV test animals avoided the dark side for a longer latency period than AII-injected ~nim~l~, or CSF-injected control ~nim~lc Materials and Methods:
Hippocampal AT4 receptor studies:
Hippocampus was from 4-month old male guinea pigs following decapiLaLion.
The tissue was homogenized in 40 volumes of hypotonic buffer co~ g 50mM
Tris, pH7.4 and 5mM EDTA, and spun at 1000g for 10 min. The supernatant was removed and recel~Llir~lged at 40,000g for 30 min. The pellet was rehomogenized in hypotonic buffer and lt;cenL~iruged. The 40,000g pellet was homogeni7ed in isotonic buffer (50mM Tris, pH7.4, 5mM EDTA, 150mM NaCl, 20mM bestatin, 50mM
Plummer's inhibitor, 100mM PMSF, and 0.1% heat treated BSA) and ~ect"L~i~uged a final time at 40,000 x g. The pellet was resuspended at a concellLl~lion of 2.5mg protein/ml as determined by the Lowry protein assay. Binding assays, which totaled 250ml, co"lailled 10ml l25I-AIV ligand (sp. act-2176 Ci/mmol), 10ml tissue homogenate, 10ml unlabeled peptide (if employed), and the rçm~indçr isotonic buffer.
Tnr.ub~tions were carried out for 2 h at 37C. Plelilllill~y c,.~ "ents demonstrated that ;l.r..lbAlion for 1 h at 37C was n~cç.cc,..y for equilibrium to be reached and that binding was stable for at least 4 h. At that time less than 10% of the l25I-AIV was shown to be by HPLC analysis. Saturation isoLl,~""s were developed using 12 concc;nl,~lions of 125I-AIV in duplir~te and inrl-lded total and nonspecir,c binding [+lOOnM AIVl. Co~.pet;l;on curves were developed using 500,000cpm/tube (0.6nM) of 125I-AIV and varying llnl~beled peptide (10~M to 10-1lM~ in half-log dilutions (Dup 753), CGP42112A: 10~Mto 10-11M).
Autoradiographic studies:
Autoradiographic analysis of Hippocampus binding was carried out using 20mM tissue sections mounted on slides. Slices were initially prPincub~ted in isotonic ~vo 94/00492 2 1 3 9 1 0 5 Pcr/us93/06038 buffer for 30 min at room temperature, then incllbated in labeled ligand (0.6nM) for 2 h, rinsed, dried, and exposed to X-ray film as previously described.

Isolation~ Purification. and Characterization of the AIV An~iotçn~in~e Enz,vme AIV An~iotPn.~in~e:
The results of studies conduced in Examples 1-3, above, with bovine adrenal cortex inrlic~te that a high affinity peptidase (Km=3nM) is present in these plepal~ions that is capable of catalyzing hydrolysis of AII or AIII to AIV.
Hydrolytic conversion of AII (or AIII) to AIV may result from the action of an AIV-specific aminoendopeptidase, capable of hydrolyzing an arginyl-valinyl peptide bond (between positions #2 and #3 in AII; Figure 1) in an angiotensin termed herein AIV-angiotçn~in~e. (Alternatively, the conversion of AIII to AIV may result fromthe action of no~ ,ec;r.c proteases but these en~.yllles may also cleave all angiotensins at sites other than the AII R2-V3, and are not termed herein AIV angiot-Pn~in~e.). In either case, cleavage of the AII Arg2-Val3 peptide bond in AI, AII, or AIII generates AIV.
Considering the important evolutionary conservation of the AT4 receptor and AIV ligand, and their most signifir~nt physiological roles, it is most likely that certain tissues and cells possess a specific AIV angiol~ e el.~yllle(s), i.e., that cleaves AI, AII, and Am in an çffit iPnt manner to permit re~ t~ble formation of AIV. The AIV angiotçn~in~e enzyme may be id~ntifietl isolated, and purified using the experimental approaches described below, in the Materials and Methods, in colllbination with the assays described in the Examples above (see Example 1). Data pl~;sellled herein indicate that AII and AIII are excellent and specific inhibitors of 125I AIV formation from 125I-AI.
Materials and Methods:
Ex~o~lilll~llL #1. Formation of AIV Ligand From AIV Precursors in Circulation.
12sI-labeled angiotens~s (107 dpm) - AI, AII, or Am, and tetr~dec~replide can be injected into the carotid artery of a guinea pig and blood s~mrlçs (50~11) can be collected at 30-sec or 1-min. intervals from a second cannula in the femoral artery into 100~11 of 20% TCA for 10 min. Samples may be analyzed by reverse-phase HPLC
ili7.ing methods that have been reported previously (47). The data are analyzed to deterrnine the rate of formation of AIV ligand from potential AIV precursors.
Ex~elilllt;llL #2: Formation of AIV from Precursors via Action of Adrenal Enzymes.
Guinea pig adrenals were excised and homogenized in a Krebs-Ringer buffer co.... ...l;.il~ g the full comrlçm-Pnt of ions (as above in Example 1). After a low speed WO 94/00492 PCI /US93/0603~
21391û~

spin at 500g for 10min. to remove whole cells and nuclei, the supellla~ is centrifuged at 40,000g for 30 min. The supernatant is recellllirllged at lOO,OOOg for 90 min. yielding both a soluble (lOO,OOOg sup~llla~ ) and a microsomal (lOO,OOOgpellet) fraction. The 40,000xg pellet is rehomogenized and fractionated on a discontinuous sucrose fractionated gradient (0.4M-1.2M sucrose, in 0.2M steps).
The lll~lllbl~1es at the 0.8M to 1.M and lM to 1.2M interfaces can be collected and cGllll,hled, resuspended in a 10X excess of Krebs buffer. The lllelll~lanes were then centrifuged at 40,000 x g for 30 min. After a final resuspension in Krebs buffer and centrifugation at 40,000 x g for 30 min., the final plasma lll~;lllbl~e fraction is ready for the assay. Soluble, lllelllbl~ e, and microsomal fractions may be in~ub~ted at various protein concc;llLl~lions and times at 37C with 106 cpm of 125I-AI, AII, AIII, and tetradecapeptide. Conditions were chosen (as above) to yield less than 10% total pre~iul~or hydrolysis thus assuring that Colllp~isons of conversion rates is carried out under initial rate conrii~ionc The reaction is termin~ted with 20% TCA and the products were evaluated by reverse-phase HPLC. The assay may also be useful for identifying AIV angiotencin~ce enzyme in clrollla~ographic and other SDS-PAGE
fractions isolated from adrenal, plasma, neural, and other tissues and bodily fluids.
E~ c;lhllt;ll~ #3: Characterization of AIV-Specific Angiotçncin~ce.
If guinea pig adrenal tissue (as expected) possesses an AIV angiot~ncin~c~ the specificity of the el~yllle(s), its activity on various substrates, and metal ion requilt;lll~;llL~ can be established by inr.ubating plt;p~uaLions of the icol~ted enzyme with angiotensins (e.g., in the presence of ;l~ Ol~ of nons~,ec;rlc proteases), and followed by ~...;n~lion of the hydrolytic products on reverse-phase HPLC. The sequence of the hydrolytic products may be de~ellllined by automated amino acid 25 sequencing. Tncubation conditions with varying concellLl~;ons of the angiotensin substrate were used to develop data for double reciprocal plots thus allowing the affinity of el~yllle(s) for the dirrel~ angiotensins to be detellll.ned. Next, colllpeL;l;on studies can be undertaken using various angiotensin analogues and ul~ela~ed peptides in order to establish the structural lt;~lu;lt;lll~ s of the AIV
30 angiotenc;~ce el~yllle(s). Finally, the ability of numerous divalent ions to activate AIV angiote-ncin~ce can be moni~ored. These c,~elilll~llLs can be carried out with AIV angiotçncin~ce el~yllles that have been EDTA-~Ll;pped and the EDTA/Me++
removed by dialysis.

-`'094/00492 2139105 PCI/US93/06038 CITATIONS
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4. J.W. Harding and D. Felix, Brain Res. 410, 130 (1987) 5. D. Regoli, B. Riniker, and H. Brunner, Biochem. Pharmacol. 12, 637-646 (1963) [see also #2, #9, #10]
6. F.M. Bumpus, P.A. Khairallah, K. Arakawo, I.H. Page and R.R. Smeby, Biochem. Biophys. Acta 46, 38-44 (1961) 7. D. Regoli, W.K. Park and F. Rioux, Pharmacol. Reviews 26, 69-123 (1974) [see also #6, #10, #3]

8. Bennett, J.P. and Snyder, S.H., Angiotensin II binding to .. A.. ~ n braines, J. Biol. Chem. 251, 7423-7430, (1976). Colossman, H., Bankal A., and Catt K.J. Plopcllies of angiotensin II receptors in the bovine and rat adrenal cortex. J. BioL Chem. 249, 825-834 (1974)

9. Fitsimons, J.T. J. Physiol Lond. 214, 295-303 (1971).

10. Tonnaer, J.A., Weigant, V.M., Degong, W. and DeWeid, D., Brain Res. 236, 417-428 (1982).

11. Siemens, I.R., Swanson, O.N., Flaharty, S.J., and Harding, J.W., J.
Neurochem 57, 690-700 (1991)

12. T. Kono, F. Ikeda, F. Oseko, Y. Ohmori, R. Nakano, H. Muranaka, A.
T~ni~1chi H. Imura, M.C. Khosla and F.M. Bumpus, Acta endocr. 99, 577-584 (1982).

13. Kono, T. et al., Acta Endocr. 109, 249-253 (1985)

14. R.L. Haberl, P.J. Decker and K.M. Ejnhaupl, Circ. Res. 68, 1621-1627 (1991)

15. J.J. Brazko, J. Wlasienko, W. Koziolkiewicz, A. Janecka and K. Wisniewski, Brain Res. 542, 49-54 (1991)

16. J.J. Brazko, G. Kuply~,t;w~ki, B. Witczuk and K. Wisniewski, Neurosci 27, 777-783 (1988)

17. J.J. Brazko, K. Wisniewski, G. Kllply~c;wski and B. Witczuk, Beh~v. Brain Res. 25, 195-203 (1987)

18. P.F. Semple, A.S. Boyd, P.M. Dawes and J.J. Morton, Circ. Res. 39, 671-678 (1976).

19. B. Blumberg, A.L., et al., (1977) Circ. Res 41, 154-158 (1977).

WO 94/00492 39~5 -80- PCI`/US93/0603

20. J.P. Bennett and S.H. Snyder, Eur. J. Pharmacol. 67, 11 (1980).

21. Kumar, S. Keegen, A., Erroi, A., West, D. Kumar P., and Gaffney, J., Prog.
App. Microcirc. 4, 54-75 (1984).

22. Fernandez, L.A., Twickler, J. and Mead, A. Lab. Clin Med. 105, 141-145 (1985)

23. Patel, J.W. et al., Amer. J. Physiol. 256, 987-993 (1989).

24. King, S.J., Beck, J.C., Harding, J.W., and Hosick, H.L., Abstract Amer. Soc.
Cell Biol. (1986)

25. Baker, K.M. and Aceto, J.F., Am J. Physiol. 259, H610-H618 (1990).
10 26. Baker, K.M., Chernim, M.I., Wixson, S.K., and Aceto, J.F., Am. J. Physiol., 259, H324-H332 (1990).
27. Y~m~lr.hi, T., Naito, Z., Stoner, G.D., Franco-Saanz, R. and Mulrow, P.J., Hypertension 16, 635-641 (1990).
28. Carpenter, G., King, L. Jr., and Cohen, S., J. Biol. Chem. 254, 4884-4891 (1979).
29. Munson, P.J., and Rodbard, D., Anal. Biochem. 107, 220-239 (1980).
30. FrPi~sm-lth~ M., Casey, P.J., and Gilman, A.G. FASEB J. 3, 2125-2131 (1989).
31. Brown, A.M. and Birnbauer, L., Am. J. Physiol. 254, H401-H410 (1988).
32. Schulz, S., Chinkers, M., and Garbers, D.L., FASEB J. 3, 2026-2035 (1989).
33. Nishibe, S. Wahl, M.I., Hernandez-Sotomayor, S.M.T., Tonks, N.K., Rhee, S.G., and C~l,t;.,ler, G., Science 250, 1253-1256 (1990).
34. P~n-liPII~ A., Bequinot, L., Vin~.çn~ini, L.M., and Meldotesi, J., TIPS 10, 411-414 (1989).
35. Cohen, S., Carpenter, G., and King, L. Jr., J. Biol. Chem., 255, 4834-4842 (1980).
36. Pang, T.P., Wang, J.K.T., Valtork, F., RP.ntPn~ti F., and Coreengard, P., PNAS 85, 762-766 (1988) 37. Dean, N.M. and Moyer, J.D., J. Biol. Chem. 250, 493-500 (1988).
38. Wright, J.W., Jensen, L.L., Roberts, K.A., Sardinia, M.F., and Harding, J.W, Am. J. P~ysiol. 257, R1551-R1557 (1989).
39. Gill, G.N., Ill, C.R., and Simonian, M.H., Proc. Nat. Aca~ Sci. 74, 5569-5573 (1977).
40. Livett, B.G., Mit~`hpllhill K.I., and Dean, D.M., "In vitro methods for studying secretion", 171-176 (1987b).

'`'O 94/00492 , , PCr/US93/06038 41. Livett, B.G., Marley, P.D., ~it~ llhill, K.I., Wan, D.C.C., and White, T.D., "In vitro methods for studying secretion", 177-204 (1987a).
42. Aceto, J.F. and Baker, K.H., Am J. Physiol. 258, H806-H813 (1990).
43. Men~lel~ohn, F.A.D. et al., Proc. Natl. Acad. Sci. USA 81, 1575-1579 (1984).
44. Speth, R.C. et al. In: J.W. Harding et al. (Eds) "Angiotensin and Blood Pressure Re~ tion", Acad. Press, S.D., CA. p. 1-3 (1988).
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SEQUENCE LISTING
(l)GENERAL INFORMATION:
(i) APPLICANT:Harding, J.W.
(ii)TITLE OF INVENTION:"~n~int~ncin IV Peptides and Receptor"
(iii)NUMBER OF SEQUENCES:6 (iv)CORRESPONDENCE ADDRESS:
(A)ADDRESSEE:Ch. .~ .., O'Connor, Johnson and Kinrln.o.cc (B)STREET:2800 Pacific First Center, 1420 Fifth Avenue (C)CITY: Seattle (D)STATE W~
(E)COUNTRY:USA
(F)ZIP:98101-2347 (v)COMPUTER READABLE FORM:
(A)MEDIUM mE:Diskette-5.25 inch, 1.2Mb storage 1 5 (B)COMPUTER:IBM PC/386 C~
(C)OPERATING SYSTEM:MS-DOS 4.01 (D)SOFTWARE:Word for Windows-t (vi)CURRENT APPLICATION DATA:
(A)APPLICATION NUMBER:
(B)FLING DATE:
(C)CLASSIFICATION:
(vii)PRIOR APPLICATION DATA:
(A)APPLICATION NUMBER:none (B)FILING DATE:none (viii)ATTORNEY/AGENT INFORMATION:
(A)NAME:S~ln~lCn n,John,S.
(B)REGISTRATION NUMBER:34,446 (C)REFERENCE/DOCKET NUMBER:WSUR-1-6263 (ix)TELECOMMUNICATION INFORMATION
(A)TELEPHONE:1-206-682-8100, 1-206-224-0727 (direct) (B)TELEFAX: 1-206-224-0779 (C)TELEX:4938023 (2)INFORMATION FOR SEQ ID NO: 1:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH:14 amino acids ""O 94/00492 2 1 3 9 1 0 5 PCr/US93/06038 (13)TYPE:amino acid (C)STRANDEDNESS:single (D)TOPOLOGY:linear (ii)MOLECULE TYPE:peptide - 5 (A)DESCRlPTION ~n~;ot~ ~r,~
(ix)SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Asp Arg Val Tyr Ile His Pro Phe His Leu Val Ile His Ser (3)INFORMATION FOR SEQ ID NO:2:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH: 10 amino acids (B)TYPE:amino acid (C)STRANDEDNESS:single 1 5 (D)TOPOLOGY:linear (ii)MOLECULE TYPF pepti-l~
(A)DESCRIPTION:~ngi~t.oncin I
(ix)SEQUENCE DESCRIPTION: SEQ ID NO:2:
Asp Arg Val Tyr Ile His Pro Phe His Leu (4)INFORMATION FOR SEQ ID NO:3:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH:9 amino acids (B)mE:amino acid (C)STRANDEDNESS:single (D)TOPOLOGY:linear (ii)MOLECULE TYPF ~pti.l.~
(A)DESCRIPTION: [des-Asp] ~- jr: I
(ix)SEQUENCE DESCRIPTION: SEQ ID NO:3:
Arg Val Tyr Ile His Pro Phe His Leu (5)1NFORMATION FOR SEQ ID NO:4:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH:8 amino acids WO 94/00492 - PCT/US93/0603~

(B)TYPE:amino acid (C)STRANDEDNESS:single (D)TOPOLOGY:linear (ii)MOLECULE TYPE:peptide (A)DESCRI~ION: An~
(ix)SEQUENCE DESCRIPTION: SEQ ID NO:4:
Asp Arg Val Tyr Ile His Pro Phe (6)rNFORMATION FOR SEQ ID NO:5:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH:7 amino acids (B)TYPE:amino acid (C)STRANDEDNESS:single 1 5 (D)TOPOLOGY:linear (ii)MOLECULE TYPE:peptide (A)DESCRI~I ION:An~int~ncin nI
(ix)SEQUENCE DESCRIPTION: SEQ ID NO:5:
Arg Val Tyr Ile His Pro Phe (7)INFORMATION FOR SEQ ID NO:6:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH:6 amino acids (B)TYPE:amino acid (C)STRANDEDNESS:single (D)TOPOLOGY:linear (ii)MOLECULE TYPF.-p~tid~
(A)DESCRIPIlON:A~ IV
(ix)SEQUENCE DESCRIPTION: SEQ ID NO:6:
Val Tyr Ile His Pro Phe

Claims (32)

What is claimed is:

1. A substantially purified angiotensin AT4 receptor having a binding affinity with a Kd of below 3 x 10-6M for an AIV ligand having the sequence VYIHPF and having a binding affinity with a Kd greater than 1 x 10-6M for AII orAIII.

2. An AT4 receptor or fragment thereof of Claim 1 which comprises a polypeptide having a molecular size of 60kD to 200kD on SDS-PAGE.

3. An AT4 receptor of Claim 1, wherein the receptor exhibits a binding affinity to VYIHPF with a Kd less than 3 x 10-8M.

4. An AT4 receptor binding site polypeptide comprising a first subdomain capable of binding an AIV ligand having a penultimate N-terminal norleucine residue, and a second subdomain capable of binding a C-terminal region of said AIV ligand, wherein the first subdomain comprises both a hydrophobic pocket conforming closely to the space filled by norleucine and a negatively charged amino acid side chaincapable of electrostatic interaction with the primary amine group of the norleucine, and wherein binding of an amino acid at the second subdomain alters the binding affinity at the first subdomain.

5. An AIV ligand capable of binding the AT4 receptor of Claim 1 with a binding affinity having a Kd of below 3 x 10-6M, said ligand comprising a compound of the formula:
R1R2R3X, wherein R1 is a substituted or unsubstituted amino acid residue having a neutral or positively charged aliphatic side chain Z1, said amino acid being selected from among V, I, L, A, G, F, P, M, K, norvaline, norleucine, and ornithine, R2 is a substituted or unsubstituted neutral nonpolar amino acid residue selected from among Y, W, N, Q, F or C, R3 is a substituted or unsubstituted neutral polar amino acid residue selected from among G, A, V, I, L, F, P, or M, and X is nothing, R4, R4-R5, or R4-R5-R6, wherein R4 is a substituted or unsubstituted basic amino acid residue selected from the group consisting of K, R and H, R5 is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, V, I, L, F, P, and M, and R6 is a substituted or unsubstituted neutral polar amino acid residue selected from the group consisting of G, A, V, I, L, F, P, M, and polyamino acid residues containing one or amino acidresidues which do not prevent binding of the AIV ligand with the AT4 receptor;
with the proviso that R1 can not be V when R2 is Y, R3 is I, R4 is H, R5 is P
and R6 is F.

6. An AIV ligand of Claim 5 wherein Z1 comprises an aliphatic chain of 4 carbon atoms in length.

7. An AIV ligand of Claim 5 wherein the amino acid residues are linked by peptidic linkages.

8. An AIV ligand of Claim 5 which comprises one or more non-peptidic linkages between adjacent amino acid residues.

9. An AIV ligand of Claim 5 which in which one or more of R4, R5, and R6 comprises a D-amino acid residue.

10. An AIV ligand of Claim 5 comprising an N-terminal sequence of VYIHP, VYIH, VYI, KYIHPF, KYIHP, KYIH or KYI.

11. An AIV ligand of Claim 5 comprising a first N-terminal L-amino acid residue having a flexible aliphatic carbon side chain and a primary amine, and a second L-amino acid residue having a phenolic side chain, wherein the first and the second amino acid residues are chemically bonded through a carbon nitrogen bond that comprises a planar or non-planar rigid or non-rigid bond having a bond length substantially equivalent to a carbonyl bond.

12. An AIV ligand of Claim 11, wherein the flexible aliphatic side chain comprises NH3(CH2)x- or CH3(CH2)y-, wherein x and y are integers from 1 to 10.

13. An AIV ligand of Claim 11, wherein x or y is 3 or 4.

14. An AIV ligand of Claim 11, wherein R1 is selected from the group consisting of norleucine, norvaline, ornithine, lysine.

15. An AIV ligand of Claim 11, wherein R2 is tyrosine.

16. An AIV ligand of Claim 12, selected from the group consisting of NorLeuYIHPF, NorValYIHPF, OrnYIHPF, and KYIHPF.

17. An AIV antagonist or agonist capable of binding the receptor or fragment of Claim 1 with a Kd of below 3 x 10-6M.

18. A method of inhibiting proliferation of a vascular smooth muscle cell in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.

19. A method of inducing proliferation of an endothelial cell in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.

20. A method of inducing increased production of an endothelial cell relaxing factor in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.

21. A method of increasing renal blood flow in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.

22. A method of inhibiting an activity induced by AII or AIII in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.

23. A method of enhancing AIV activity in a host in need thereof, comprising administering to the host a therapeutically effective dosage of an AIV
agonist ligand of Claim 5.

24. A method of enhancing memory or learning in a host in need thereof comprising administering to the host a therapeutically effective dosage of an AIV
agonist ligand of Claim 5.

25. A method of inhibiting AIV activity in a host in need thereof, comprising administering to the host a therapeutically effective dosage of an AIV
antagonist ligand of Claim 5.

26. A method of inhibiting AII-mediated aldosterone release from an adrenal cortical cell in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.

27. A method of altering catecholamine release from an adrenal medullary cell in an animal host in need thereof, comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.

28. A method of potentiating cardiocyte growth in an animal host in need thereof, comprising comprising administering to said host a therapeutically effective dosage of an AIV ligand of Claim 5.

29. A method of identifying the presence of an inhibitor of AIV ligand binding to an AT4 receptor in a biological fluid, comprising the steps of:
a) adding an amount of an AIV ligand effective to produce measurable receptor binding to a first cell culture comprising an AT4 receptor to form a control mixture;
b) adding said AIV ligand and a sample of said biological fluid or fraction thereof to a second cell culture comprising AT4 receptor;
c) measuring the level of binding of said AIV ligand to the cells in the first and second cultures; and d) determining the presence of an inhibitor of AIV ligand to the AT4 receptor when the level of binding in the second culture is significantly lower than in the first culture.

30. An antibody capable of specifically binding to AIV, but not to AII or AIII.

31. A method of determining the presence or amount of AIV in a sample, comprising contacting the sample with an antibody of Claim 30 and then determining the amount AIV bound or unbound to the antibody as an indication of the presence or amount of AIV in the sample.

32. A method of isolating and substantially purifying an AT4 receptor to remove ATl and AT2 receptors, comprising the steps of:
a) selecting cells expressing an AT4 receptor;
b) preparing a membrane preparation of said cells in the presence of protease inhibitors, wherein said protease inhibitors are capable of inhibiting greater than 90% of angiotensin hydrolysis in the membrane preparation;

c) solubilizing said AT4 receptor in said membrane preparation with a zwitterionic detergent under conditions that favor solubilization of said AIV
receptor but not an AT1, or AT2 angiotensin receptor;
d) heat-treating said solubilized preparation under conditions suitable for destroying said ATI and AT2 receptors; and, e) fractionating said solubilized AT4 receptor preparation and identifying fractions capable of binding said AIV ligand, but not said AI or AIIligands.