EP0939902A2 - Auf der messung der rezeptoroligomerisation beruhendes verfahren zum testen von verbindungen, die aktivität vom g-protein gekoppelten rezeptoren beeiflussen - Google Patents

Auf der messung der rezeptoroligomerisation beruhendes verfahren zum testen von verbindungen, die aktivität vom g-protein gekoppelten rezeptoren beeiflussen

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Publication number
EP0939902A2
EP0939902A2 EP97928072A EP97928072A EP0939902A2 EP 0939902 A2 EP0939902 A2 EP 0939902A2 EP 97928072 A EP97928072 A EP 97928072A EP 97928072 A EP97928072 A EP 97928072A EP 0939902 A2 EP0939902 A2 EP 0939902A2
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European Patent Office
Prior art keywords
receptor
receptors
activity
protein
peptide
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EP97928072A
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English (en)
French (fr)
Inventor
Michel Bouvier
Michael Dennis
Terence E. Hebert
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Universite de Montreal
Biosignal Packard Inc
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Universite de Montreal
Biosignal Inc
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Publication of EP0939902A2 publication Critical patent/EP0939902A2/de
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the present invention relates to a method of assaying compounds for the ability to modulate the function of G protein-coupled receptors based on measurements of receptor oligomerization state.
  • G protein-coupled receptors are typically characterized by a 7-helix organization, whereby the receptor protein is believed to traverse the membrane seven times. They also share a common signalling mechanism, whereby signal transduction across the membrane involves intracellular transducer elements known as G proteins.
  • G proteins G protein-coupled receptors
  • GDP guanosine diphosphate
  • GTP guanosine triphosphate
  • activation of GPCRs by transmitters will induce one or another of the following effector responses: activation of adenylyl cyclase, inhibition of adenylyl cyclase or stimulation of phospholipase C activity.
  • activation of adenylyl cyclase When the effector adenylyl cyclase is either activated or inhibited it produces changes in the concentration of the molecule cyclic adenosine monophosphate (cAMP).
  • Another effector, phospholipase C causes one molecule of phosphatidylinositol- bisphosphate (PIP2) to be cleaved into one molecule each of inositol triphosphate (IP 3) and
  • IP3 calcium ions (Ca ) to be released into the cytoplasm.
  • Alterations in cellular levels of cAMP and Ca are two of the most important intracellular messages that in turn act to alter the behaviour of other target proteins in the cell.
  • GPCRs may be classified according to the type of signalling pathway they activate in cells. This occurs at the level of the G proteins, which detect and direct signals from diverse receptors to the appropriate effector-response pathway.
  • the three main groups of G proteins are: Gs-like, which mediate adenylyl cyclase activation; Gi-like, which mediate inhibition of adenylyl cyclase; and Gq-like, which mediate activation of phosphoplipase C. Since one receptor can activate many G proteins, the signal can be greatly amplified through this signal transduction pathway.
  • GPCRs A wide variety of chemical messengers involved in regulating key functions in the body act through GPCRs. These include neurotransmitters such as dopamine, acetylcholine and serotonin, hormones of the endocrine system such as somatostatin, glucagon and adrenocorticotropin, lipid mediators such as prostaglandins and leukotrienes, and immunomodulatory proteins such as interleukin-8 and monocyte-chemoattractant polypeptide.
  • the family of GPCRs also includes the receptors for light (rhodopsin), for odours (olfactory receptors) and for taste (gustatory receptors).
  • G protein-coupled receptors Over one hundred different G protein-coupled receptors have been identified in humans, and many more are expected to be discovered. All or most of these receptors are believed to utilize one of the three principal G protein-effector signalling pathways (stimulation or inhibition of adenylyl cyclase or activation of phospholipase C).
  • 5-HT 1A 5-HT 1B 5-HT 4 5-HT, Serotonin
  • G protein-coupled receptors In view of the diverse functions of G protein-coupled receptors in the human body, it is not surprising that the pharmaceutical sector has great interest in the development of new drugs which target G protein-coupled receptors for potential therapeutic applications in a wide range of human pathologies, including psychiatric disorders (depression, psychoses, bipolar disorder), metabolic disorders (diabetes, obesity, anorexia nervosa), cancer, autoimmune disorders, cardiovascular disorders, neurodegenerative disorders (Alzheimer's disease) and pain disorders.
  • the process of discovering and developing new therapeutic drugs which act on G protein- coupled receptors involves the systematic testing of drug candidate compounds in biological assay systems which contain the targeted G protein-coupled receptor in a functional state. The goal of this testing is to identify those compounds, among a very large number of candidates, which can modulate the function of the targeted G protein-coupled receptor in a predictable and therapeutically-relevant manner.
  • Most assay systems used for drug screening classify compounds into three broad categories: 1) inactive, i.e. the compounds have no effect on receptor function at relevant doses; 2) agonists, i.e. the compound mimics the natural chemical messenger by activating the receptor; and 3) antagonists, i.e. the compound inhibits receptor activation by the natural chemical messenger.
  • the ligand binding assay detects compounds that can interact with and bind to the receptor at the same site as the natural chemical messenger. This usually involves the use of radioactive derivatives of either the natural chemical messenger or of known drugs which bind to the same receptor site, and measurement of the ability of test compounds to block the binding of the radioactive drug to the targeted receptor present in a biological preparation (e.g. a tissue extract).
  • a biological preparation e.g. a tissue extract.
  • the radioligand binding assay also permits the ranking of compounds based on binding affinity, i.e. the concentration of the compound which results in occupation of half of the receptors in the preparation. In general, the lower the concentration of compound necessary to occupy half of the sites (i.e. the higher the affinity), the better the candidate.
  • Radioligand binding assays while widely employed in the first steps of drug screening, have a number of limitations, the most severe being the inability of this assay to discriminate between agonists and antagonists.
  • the functional bioassay tests the effect of the compounds on receptor activity, i.e. the ability of the receptor to transmit signals across the cell membrane to control cellular response pathways. Since G protein-coupled receptors control a wide spectrum of cellular functions, the functional bioassays used in drug screening for G protein-coupled receptors include a large variety of different tests which monitor any one of a series of biochemical or cellular processes which are under the control of receptor activity. These assays all permit the discrimination of agonists from antagonists, i.e. agonists will activate receptor signalling pathways, while antagonists will block activation of signalling pathways by receptor agonists (such as the natural chemical transmitter).
  • most functional bioassays can also rank agonist compounds based on efficacy, i.e. the maximum level of activation of the signalling pathway achieved by the agonist compound.
  • Full agonists result in full activation of the receptor- controlled process, whereas partial agonists can induce only fractional activation of the
  • adenylyl cyclase activity activation or inhibition of adenylyl cyclase activity or activation of phospholipase activity
  • modulation of post-effector signalling proteins such as kinase and phosphatase enzymes, ion channels, transcription factors, etc.
  • modulation of integrated cellular responses such as secretion (e.g. for glandular cells), contraction (e.g. for smooth muscle), electrical activity (e.g. for neurons), growth and proliferation (e.g. for endothelial cells).
  • recombinant receptors has many advantages over tissue sources, including the ability to use human receptors expressed from human genes, the facility with which large amounts of the protein can be produced, and the fact that a single receptor subtype can be tested and compared against closely related subtypes (receptor subtypes are receptors that are closely related but distinct, yet which use the same natural transmitter).
  • Bioassay systems for recombinant G protein-coupled receptors that are known in the art are based on the ability of the expressed receptor to activate endogenous signalling pathways in the host cell.
  • Early assays measured the activity of effectors (adenylyl cyclase and phospholipase C) using known biochemical assays originally used for tissue-based assays. These generally employ mammalian cell lines which have been made to express the cloned receptor DNA using techniques (eg. transfection, transformation) which are well known to and routinely practised by technicians trained in the art.
  • One example uses fluorescent dyes sensitive to the concentrations of specific ions, primarily calcium, to measure changes in the intracellular ion concentrations associated with activation of receptors coupled to Gq- phospholipase C signalling.
  • an increasing number of new assay systems involve genetic engineering of the host cell to facilitate measurement of the effector response to receptor activation.
  • gene reporter assays the gene for an enzyme that is readily assayed, such as beta-galactosidase, is inserted into the host cell genome under the control of a gene promoter element which is normally under the control of a receptor signalling pathway.
  • Receptors which activate the specific signalling pathway will activate expression of the beta-galactosidase reporter gene.
  • Measurement of the enzyme activity in a simple assay thus provides a measure of receptor activity and provides a functional bioassay to monitor the activity of compounds on the receptor.
  • a variation of this type of assay uses the yeast Saccharomyces cerevisiae as a microbial host cell to express human G protein-coupled receptors which are coupled to an endogenous yeast signalling pathway controlling the response to sex pheromones.
  • the receptor activates a yeast promoter which in turn controls the expression of a reporter enzyme (e.g. beta-galactosidase).
  • Another approach is to express the receptors in specialized cells that have endogenous respons e mechanisms that allow convenient assay of ligand activation of the receptor.
  • receptors that change cAMP levels have been cloned in melanophores (cultured pigment cells) wherein altered cAMP levels alter cellular colour, a response that is a conveniently measured response (Potenza et al. , 1992, Anal. Biochem. 206:315).
  • melanophores cultured pigment cells
  • Yet another assay strategy is based upon the ability of certain receptors to alter cellular growth.
  • Cells of the NIH 3T3 fibroblast cell line have been extensively used to evaluate the activity of a large diversity of gene products that control cell growth, and a number of receptors are able to control the activity of these cells when stimulated by individual ligands.
  • carbachol a muscarinic agonist stimulates cells transfected with certain muscarinic receptors (Gutkind et al. , 1991 , Proc. Natl. Acad. Sci. USA, 88:4703; Stephens et al. , 1993, Oncogene, 8: 19), and Norepinephrine stimulates cells transfected with certain b- adrenergic receptors (Allen et al. , 1991 , Proc. Natl. Acad. Sci. USA, 88: 11354).
  • agonist ligands several characteristics of the cells are altered, including cellular growth, loss of contact inhibition, and formation of macroscopic colonies termed foci. Proprietary methods have been developed in the art in order to facilitate detection of such foci.
  • Figure 1 shows immunoblotting of human ⁇ 2AR expressed in Sf9 cells. Crude membrane preparations (lane 1), digitonin-solubilized membrane proteins (lane 2) and affinity purified receptors (lane 3) derived from Sf9 cells expressing either c-myc tagged (lane 3) or HA-tagged
  • ⁇ 2 R were immunoblotted following SDS-PAGE using the appropriate antibody (9E10 and 12CA5, respectively).
  • the blots reveal immunoreactive bands corresponding to the expected monomeric form (43-50kDa) as well as a higher molecular weight species (85-95 kDa).
  • the right panel illustrates immunoblots of crude membrane preparations derived from Sf9 cells expressing HA-tagged ⁇ 2AR treated (lane 5) or not (lane
  • Figure 2 shows effects of various peptides and ⁇ 2AR ligands on receptor dimerization.
  • Lanes 1 and 2 c- myc (lane 1) or anti-HA (lane 2) mAbs.
  • the two immunoprecipitates were then immunoblotted with the anti-HA mAb.
  • the occurrence of dimerization between the HA- and c-myc-tagged receptors is revealed by the fact that the HA-tagged ⁇ 2AR is co- immunoprecipitated with the c-myc tagged receptor by the anti-c-myc mAb (lane 1).
  • Lanes 3 and 4 c-myc tagged ⁇ 2AR was expressed in Sf9 cells and immunoprecipitated with anti-c- myc mAb. The immunoprecipitates were then immunoblotted with either anti-HA (lane 3) or anti-c-myc or anti-c-mvc (lane 4) mAbs. Lanes 5 and 6: HA-tagged ⁇ 2AR was expressed in Sf9 cells, immunoprecipitated with anti-HA mAb and then immunoblotted with either anti-c- myc (lane 5) or anti-HA (lane 6) mAbs. These controls demonstrate the specificity of each antibody towards their respective targets. Lane 7 and 8: HA-tagged ⁇ 2 R and c-myc tagged
  • M2 muscarinic receptors were co-expressed in Sf9 cells, immunoprecipitated with either anti- HA (lane 7) or anti-c-myc (lane 8) mAbs. Immunoblotting with the anti-c-mvc mAb did not reveal the presence of a ⁇ 2AR/M2 muscarinic receptor heterodimer (lane 8). Results shown are representative of three separate experiments.
  • FIG. 3 demonstrates immunoblotting of V2-vasopressin receptors (V2-R) expressed in COS- 7 cells.
  • V2-R V2-vasopressin receptors
  • Crude membrane preparations trom COS-7 cells transiently transtected with c-mvc tagged V2-R (lane 1) or c-mvc tagged V2-R truncation mutant O-l l (lane 2) were immunoblotted with the anti-c-mvc mAb.
  • the molecular weight markets are as shown Square brackets highlight the dime ⁇ c species of both wildtype and O-l l V2 vasopressin receptors while asterisks denote the monomeric species Data are representative of three independent experiments.
  • Figure 4 shows effects of various peptides on receptor dimerization.
  • A Time course of the effect of the TM VI peptide on ⁇ 2AR dimerization
  • Membranes derived from Sf9 cells expressing ⁇ 2AR were treated at room temperature with TM VI peptide [residues 276-296:
  • Figure 5 demonstrates, in A, effects of increasing concentrations of TM VI peptide on the amount of ⁇ 2AR dimer.
  • Increasing concentrations (0-6.3 mM) of the peptide were added to purified c-mvc tagged ⁇ 2AR and the amount of dimer assessed by immunoblotting using the anti c-myc mAb (lanes 1 - 8)
  • the amount of dimer assessed by immunoblotting using the anti c-myc mAb (lanes 1 - 8)
  • In lanes 9 and 10 purified ⁇ 2AR was treated (lane 10) or not (lane 9) with the D2 TM VII peptide The data shown are representative of three distinct experiments.
  • control peptides used to determine the selectivity of the effect observed with the TM VI peptide included one derived from the C-terminal tail of the ⁇ 2AR [residues 347-358 NH 2 -LKAYGNGYSSNG-COOH] or an additional control peptide unrelated to the ⁇ 2 AR but of similar size as the TM VI peptide [NH 2 -SIQHLSTGHDHDDVDVGEQQ- COOH] were also found to be without effect on the amount of dimer (data not shown).
  • B Densitometric analyses of three experiments similar to that shown in B. The relative intensity of the dimer is expressed as percent of total receptor (monomer + dimer) immunoreactivity.
  • Inset shows superimposed densitometric scans of immunoblotted receptors which were previously treated with increasing concentrations of the TM VI peptide.
  • the monomer is denoted by M while the dimeric species is marked by D.
  • the concentration of peptide added for the curves shown was: none ( ), 0.07 mM ( — ... — ), 0.05 M ( — — ), and 1.25 mM ( ).
  • Figure 6 demonstrates effects of TM VI peptide on ⁇ 2AR stimulated adenylyl cyclase activity in Sf9 cells.
  • A Membrane preparations derived from ⁇ 2AR expressing Sf9 cells were either not treated (open circles), or treated with TM VI peptide (closed squares), control peptide TM VI Ala (closed circles), or second control peptide from TM VII of the D2 dopamine receptor (open triangles). Isoproterenol stimulated adenylyl cyclase activity was then assessed for these membranes. Data are expressed relative to the maximal stimulation obtained with the untreated membranes and represent mean +/- SEM for 8 independent experiments.
  • Figure 7 shows effects of ⁇ 2AR ligands on receptor dimerization.
  • A Time course of the effect of 1 ⁇ M isoproterenol on ⁇ 2AR dimerization.
  • Membranes derived from Sf9 cells expressing the c-myc ⁇ 2AR were treated at room temperature with 1 ⁇ M isoproterenol for 0
  • TM VI VI peptide at a concentration of 0.15 ⁇ g/ ⁇ L
  • ISO/PEP isoproterenol followed by 30 minutes with TM VI peptide
  • the TM VI data (lane 4) is reproduced from Figure 4b for comparison.
  • Figure 8 depicts effects of TM VI peptide on ⁇ 2AR expressed in mammalian cells.
  • Ltk- cells Membranes were prepared from Ltk- cells stably expressing 200 fmol of human ⁇ 2AR/mg membrane protein. Isoproterenol-stimulated adenylyl cyclase activity was then assessed in membranes treated with vehicle (open circles), TM VI peptide (closed squares), control peptide TM VI Ala (closed circles), or the D2 TM VII control peptide (open triangles) . Data are expressed relative to the maximal stimulation obtained with vehicle treated membranes and represent mean +_ SEM for 3 independent experiments. Peptides were used at a concentration of 0.15 ug/ul.
  • IP inositol phosphate
  • the abbreviation BASED is bis [ ⁇ -(4 azidosalicylamindo) ethyl] disulphide
  • 5-HT is 5-hydroxytryptamine.
  • DOI 2,5-dimethoxy-4-iodoamphetamine hydrobromide.
  • PBS phosphate buffered saline.
  • ⁇ 2AR is ⁇ 2-adrenergic receptor.
  • GPCR G protein-coupled receptor
  • GpA glycophorin A
  • HA influenza hemagglutinin.
  • TM VI transmembrane domain 6.
  • NDI nephrogenic diabetes insipidus.
  • Orphan receptors are receptors for which the natural ligands and/or biological function are uncertain or unknown.
  • the present invention resides in the discovery that certain GPCRs form oligomeric structures
  • a working example is provided, based on the human ⁇ 2 adrenergic receptor in which agonist promotes formation of oligomers, inverse agonist promotes dissociation of oligomers and a peptide derived from residues 276 - 296 of the ⁇ 2-adrenergic receptor inhibits agonist-promoted formation of oligomer and also inhibits stimulation of adenylyl cyclase activity.
  • This invention provides a method of testing compounds for activity and efficacy based on the ability of the compound to alter the monomer-multimer equilibrium.
  • This invention permits direct measurement of compound efficacy on the receptor independent of post-receptor signalling pathways, and is applicable to G protein-coupled receptors of different functional classes.
  • Methods of screening compounds for activity and efficacy based on their ability to alter association-dissociation of GPCRs are unknown in the art because GPCRs are not thought to undergo association/dissociation as part of their activity.
  • This invention can also apply to orphan GPCRs, for which ligand specificity or receptor activities are not yet determined.
  • the novel observations described in this invention indicate that receptors in the GPCR family will undergo this fundamental oligomerization process as an integral part of their activity, thereby allowing a novel method of assaying such receptors independent of knowledge regarding ligand specificity.
  • the method of this invention can also be used to test for compounds affecting the oligomerization of homo-multimers and hetero-multimers (comprised of polypeptides from different GPCR-types). For example it could be used to test for compounds that would affect the activity of hetero-multimers formed between 5HT-type receptor polypeptides and ⁇ -AR receptor polypeptides .
  • the techniques of this invention can either be correlated to receptor activity or used without such correlation. If they are so correlated for a particular type of GPCR, then assays can be conducted by determining the multimer/monomer ratio of the receptor in order to obtain an indication of its activity without having to measure that activity directly, thereby obviating time consuming and costly procedures.
  • the principal goal of all the manifestations of the assay method embodied within this invention is to measure the category of action and the efficacy of drug candidates by determining these compounds' effects upon the ratio of monomeric receptor to oligomeric receptor (dimers, trimers, homo-multimeric, hetero-multimeric, etc.)
  • the change in ratio of the relative amounts of monomer to multimer will reflect conversion of monomers to multimers or vice versa, thus providing information on the activity and efficacy of drug candidates.
  • Those compounds which promote oligomerization would be predicted to have one activity (eg. agonist or positive efficacy) while those which promote dissociation of oligomers would be predicted to demonstrate the opposite activity (eg. inverse agonists or negative efficacy).
  • the magnitude of change in ratio and/or rate of change effected by the compound would provide a measure of the compound's efficacy and/or potency in modulating receptor activity.
  • determining the relative amount of monomer to multimer eg. dimer
  • different assay systems can be designed to measure the ability of compounds to modify the ratio of monomers/multimers.
  • any procedure that permits measurement of the relative amounts of monomer and oligomer in receptor preparations eg. membranes, solubilized receptor preparations, purified receptors, etc
  • receptor preparations eg. membranes, solubilized receptor preparations, purified receptors, etc
  • a sample containing the compound to be tested or a control sample lacking the compound would be added to a suspension or solution of receptor preparation. After an incubation period, the receptor preparation would be analyzed to determine the relative amounts of monomeric and oligomeric species such that changes in the ratio produced by the test compound could be used to predict the activity and efficacy of the compound in regulating receptor function.
  • Immunological methods can be used to measure compound efficacy.
  • differential epitope tagging can be used in combination with differential co-immunoprecipitation to demonstrate the formation or absence of multimeric subunit aggregation.
  • immunological techniques can be used to purify and identify the presence of each subunit in a multimer. If the complex is made up of two or more identical subunits (eg. homodimer or homotrimer), each subunit is treated as if it is unique, such that the subunits bear tags in proportion to the number of units in the multimer.
  • the complex is a homodimer
  • one-half of the cDNA will be tagged with tag A and the other-half will be t agged with tag B.
  • the resulting dimers will form between A-A, AB, and BB subunits, but will be observable by their migration in the SDS-PAGE gel, relative to the individual units. These will be visualized by immunoblotting with either or both types of anti-A MAbs or anti-B
  • each set comprises cDNA encoding one subunit of a receptor and one unique immunologic tag, one set for each subunit;
  • test compound add test compound to the receptor preparation; 5) immunoprecipitate the receptors using anti-tag MAbs, one per unique tag;
  • An immunological method for measuring monomer/oligomer ratio entails separating monomers and oligomers based on size and measurement of relative amounts of each using reporter systems. In this embodiment the following steps would be followed:
  • receptor cDNA would be modified such that when expressed the expressed receptor would be tagged with the epitope for a monoclonal antibody: this expression would be performed in a heterologous system (eg. baculovirus-insect cell system);
  • membranes or pure receptor would be solubilized in SDS sample buffer and components separated by size on SDS-polacrylamide gels;
  • alternate means of separating monomeric and oligomeric receptor species by size can be used: eg. gel filtration, ultracentrifugation or others followed by antibody detection of different size forms and determination of ratio of monomeric to oligomeric species.
  • Alternate means of labelling the receptor could entail labelling the 5 receptor with some reporter permitting specific detection of the receptor (eg. fluorescent label specifically incorporated into the receptor protein which can be quantified following size separation of monomeric and oligomeric species.
  • association of monomers into oligomeric receptor comp lexes o can be measured directly using Fluorescence Resonance Energy Transfer, involving use of two different fluorophores with distinct excitation and emission spectra, where the emission spectrum of the first fluor overlaps with excitation spectrum of the second fluor.
  • Two separate preparations of receptor would be labelled with one or the other fluor and these labelled receptor preparations would be reconstituted together in solution or in phospholipid vesicles. 5 The mixture would then be irradiated at the excitation wavelength of the first fluor.
  • Monomers would show major emission and emission wavelength for the first fluor. Oligomers would show increased emission at the emission wavelength of the second fluor due to close proximity of the two fluors and energy transfer from the first to the second fluor.
  • the ratio of emission intensities at the emission wavelengths for the first and second fluors would 0 provide a measure of the relative amounts of monomeric (no energy transfer) and the oligomeric receptor species.
  • Compounds which modify the ratio of monomeric and oligomeric species of the receptor will also modify the ratio of emission intensities at the two emission wavelengths and permit prediction of activity and efficacy of the compound in regulating receptor activity .
  • Modifications to this Fluorescence Resonance Energy Transfer method can be made by using receptors tagged with different epitopes and two corresponding monoclonal antibodies labelled with first and second fluors.
  • two receptor populations tag 1 and tag 2 in the same preparation (by co-expression of two receptors in insect cells or mammalian lines; or by separate expression and reconstitution into single preparation) are incubated with anti-tag 1 labelled widi fluor 1 , and anti-tag 2 labelled with fluor 2.
  • Monomers will n ot show energy transfer between fluors 1 and 2 on different receptor monomers, whereas oligomers will bring two receptor-bound antibodies into proximity and permit energy transfer, measured as an increase in emission intensity at the emission wavelength of fluor-2.
  • Compounds would be added to the mixture and tested for their abilities to promote receptor oligomerization or dissociation of oligomers into monomers, and this information would permit prediction of compound activity and efficacy in regulating receptor function.
  • the specifics of assessment assays for test compounds would thus involve the following steps : adding aqueous solution containing the test compound to be evaluated to solution containing a GPCR preparation (tissue, cell or extract); adding agonist to the same solution; measuring the response to agonist by means of an assay as described above; comparing the magnitude of the response to agonist in the presence of the peptide or peptidomimetic compound to that of the response in the absence of test molecule under otherwise identical conditions. Decrease in agonist-induced response in the presence of peptide or peptidomimetic compound indicates antagonist activity .
  • Activity of the test compound can be further characterized by testing: varying the concentrations of test compound against a fixed concentration of agonist to determine the potency of the antagonist-like test compound and then varying the concentration of the agonist with fixed test compound concentration to determine competitive versus non-competitive action. Finally, measuring the effect of test compound on progressively more distantly-related receptors can be performed in order to determine selectivity .
  • Activity of the test compounds can also be assessed by measuring the compound's effect on spontaneous receptor activity (i.e. , basal activity in absence of added agonist). In this case, the same assay systems can be used but without agonist, and the decrease in receptor activity in presence of the test compound is measured.
  • Peptides were synthesized on solid-phase supports using f-moc chemistry (Merrifield, R.B. , Rec Prog. Hormone Res. 23:451-482, 1967; Stewart. J. and Young, J. , Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Illinois, 1984) on a BioLynx 4175 manual peptide synthesizer (LKB). Peptides were solubilized in the following buffer:
  • membrane preparations from mammalian or Sf9 cells infected with recombinant baculovirus expressing human ⁇ 2AR were treated with increasing concentrations of the different peptides at room temperatures and for various times as indicated below.
  • membrane preparations from mammalian or Sf9 cells or affinity purified receptors derived from Sf9 cells expressing c- myc tagged ⁇ 2AR were treated at increasing concentrations of the different peptides at room temperature for various times as indicated (see results). Samples were then run on SDS-PAGE and then transferred to nitrocellulose.
  • membrane preparations were also treated with either 10 wM timolol or 1 ⁇ M isoproterenol instead of, or in addition to the different peptides.
  • Peptide antagonist activity was assessed by assaying adenylyl cyclase activity. In these assays, membranes were also used to determine the effect of various peptides on the ability of the ⁇ 2AR to stimulate adenylyl cyclase activity described below.
  • baculoviruses encoding the c-myc or hemaglutinin (HA) tagged wildtype human ⁇ 2 ⁇ R, the c-myc tagged human M 2 muscarinic receptor and c-myc tagged D, dopamine receptor (c-myc ⁇ 2AR and HA ⁇ AR, c-myr M 2 -R, and c-myc D,-R respectively) were constructed as described (Mouillac, B., et al. , J. Biol. Chem., 267:21733-21737, 1992). Briefly, HA (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) and c-myc
  • Sf9 cells were maintained at 27 C in serum-supplemented [10% fetal bovine serum (FBS) v/v] Grace's insect medium (Gibco-BRL) with gentamycin and fungizone. Cells were grown either as monolayers in T flasks or in suspension in spinner bottles supplemented with pluronic acid to prevent cell taring due to agitation. Cells were infected at log phase at a density of 1 x 10 cells per ml for 48 h.
  • FBS fetal bovine serum
  • Gibco-BRL Grace's insect medium
  • CHW and LTK cell lines with and without stably transfected ⁇ 2AR were maintained as described (34).
  • Cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with L-glutamate, 10% FBS, gentamycin and fungizone.
  • DMEM Dulbecco's modified eagle medium
  • Transfected CHW cells expressed 10 5 pmol receptor/mg protein while transfected LTK cells expressed 200 fmol receptor/mg protein.
  • Stably transfected cell lines were grown in the presence of 150 ⁇ g/ml G418.
  • V2 vasopressin receptors For transient expression of V2 vasopressin receptors the following procedures were followed. COS-7 cells were maintained in supplemented DMEM as described above. Genomic DNA for the V2 vasopressin receptor was isolated from nephrogenic diabetes insipidus (NDI) patients or unaffected individuals, subcloned into a construct containing a c-myc epitope tag and ligated into a mammalian expression vector, pBC12BI (Cullen, B.R, Meth. EnzymoL , 152:684-704, 1987). Using DEAE-dextran, COS-7 cells were transiently transfected with the expression vector encoding either wildtype V2 vasopressin receptor, a truncation mutant O-l l or with vector alone for 48 hours.
  • NDI nephrogenic diabetes insipidus
  • Membranes were prepared as follows and washed. Sf9 or mammalian cells were washed twice with ice-cold PBS. The cells were then disrupted by homogenization with a polytron in 10 ml of ice-cold buffer containing 5 mM Tris-HCl, pH 7.4, 2 mM EDTA (plus a protease inhibitor cocktail consisting of 5 mg/ml leupeptin, 10 mg/ml benzamidine and 5 mg/ml soybean trypsin inhibitor). Lysates were centrifuged at 500 x g for 5 minutes at 4 C, the pellets homogenized as before, spun again and the supernatants were pooled. The supernatant was then centrifuged at 45,000 x g for 20 minutes and the pellets washed twice in the same buffer. In some cases receptors were then solubilized in 2% digitonin or 0.3 %
  • N-dodecyl- -D-maltoside and purified by affinity chromatography on alprenolol-sepharose as or by immunoprecipitation as described below.
  • Solubilized receptors were affinity purified by alprenolol-sepharose chromatography as described (Mouillac, B. , et al. , J. Biol. Cem. , 267:21733-21737, 1992; Shorr, R.G.L. , et al., J. Biol. Chem. , 256:5820-5826, 1981).
  • the affinity purified preparations were concentrated using Centriprep and Centricon cartridges (Amicon) and the amount of ⁇ 2AR
  • 125 in each sample was determined in soluble [ I]CYP radioligand binding assays as described (Mouillac, et al., 1981, supra). Purified receptors were desalted on Sephadex G-
  • Tagged ⁇ 2ARs were immunoprecipitated with either a mouse anti-c-myc monoclonal antibody (9E10; Evan, G.I. , et al. , Mol. Cell. Biol. , 5:3610-3616, 1985) or a mouse anti- hemagglutinin monoclonal antibody (12CA5; Nimar, H.L. , et al. , Proc. Natl. Acad. Sci. USA, 80:4949-4953, 1983) as described previously (Mouillac, et al., 1981, supra).
  • a mouse anti-c-myc monoclonal antibody (9E10; Evan, G.I. , et al. , Mol. Cell. Biol. , 5:3610-3616, 1985
  • a mouse anti- hemagglutinin monoclonal antibody (12CA5; Nimar, H.L. , et al. , Proc. Natl. Acad. Sci. USA,
  • Membrane preparations from Sf9 or mammalian cells or in some cases affinity-purified or immunoprecipitated ⁇ 2AR were prepared for non-reducing SDS-PAGE on 10% slab gels as described previously (Laemmli, U.K., Nature, 227:680-686, 1970). In the case of the
  • V2 vasopressin receptors reducing SDS-PAGE was performed.
  • gels were transferred to nitrocellulose and blotted with either the mouse anti-c-myc monoclonal antibody (9E10), the anti-hemagglutinin monoclonal antibody (12CA5) at dilutions of 1 : 1000 or in the case of mammalian cells expressing the ⁇ 2 R, a polyclonal rabbit anti- antiserum raised against a peptide from the C-terminal region of the ⁇ 2AR at a dilution of 1 :2000.
  • Immunoblots against the anti-c-myc or anti-HA antibodies were revealed using a goat anti-mouse alkaline phosphatase-coupled second antibody (GIBCO- BRL) or a chemiluminescent substrate for a horseradish peroxidase coupled second antibody (Renaissance, NEN Dupont).
  • GEBCO- BRL goat anti-mouse alkaline phosphatase-coupled second antibody
  • Renaissance, NEN Dupont horseradish peroxidase coupled second antibody
  • For the experiments performed using mammalian cells expressing the ⁇ 2AR western blots were developed using a chemiluminescent substrate for goat anti-rabbit coupled horseradish peroxidase antisera (Sigma).
  • blots were scanned by laser densitometry (Pharmacia-LKB Ultrascan).
  • Receptor quantification and adenylyl cvclase assay 125 Receptor number was calculated from saturation binding experiments using [ I] cyanopindolol (CYP) as the radioligand (Bouvier et al. , Mol. Pharmacol., 267:7-19,
  • Adenylyl cyclase activity was assayed by the method of Salomon et al., (Anal. Biochem. , 58:541-548, 1974). Membranes were prepared and washed as described above. Again 10 ⁇ L of membranes (3-5 ⁇ g of protein) were used in a total volume of 50 ⁇ L. In some experiments, the peptides or the buffer used to solubilize them were added to the enzyme assay mix. Enzyme activities were determined in the presence of 1 nM to 100 ⁇ M isoproterenol, 100 ⁇ M forskolin or 10 mM NaF. Data were calculated as pmoles cAMP produced/min/mg protein and were analyzed by least squares regression using SigmaPlot 4.17 (Jandel Scientific).
  • the dimer which was readily observed in membrane preparations, was also detected in digitonin-solubilized receptors (lane 2) and following affinity purification of receptors on alprenolol-sepharose (lane 3).
  • lanes 4 and 5 when whole cells expressing the ⁇ 2AR were treated with the membrane permeant cross-linking agent BASED, the dimer to monomer ratio as assessed by immunoblotting was increased by two-fold. This suggests that the dimer is already present before cell fractionation and that crosslinking stabilizes this form of the receptor. therefore, the dimeric species does not represent an artifact of membrane preparation or solubilization. Identical results were obtained when membranes were solubilized with 0.3 % N-dodecyl- ⁇ -D-maltoside instead of digitonin (data not shown)
  • blotting of the anti-HA immunoprecipitate revealed both the 45 kDa and the 90 kDa forms of the receptor.
  • the ⁇ 2AR could also be detected by the anti-HA mAb in the c-myc immunoprecipitate of co-expressed receptors but the dimer then represented the predominant form (lane 1 ) .
  • V2 vasopressin receptors are also dimeric.
  • the vasopressin receptor is critical for regulation of water retention in the kidney. Recently, several mutations of this receptor have been linked to congenital nephrogenic diabetes insipidus (NDI, Bichet, D.G. , et al. , Am J. Hum. Genet. , 55:278-286, 1994).
  • NDI congenital nephrogenic diabetes insipidus
  • TM VI peptide The functional significance for receptor dimerization is suggested by the inhibitory action of the TM VI peptide on receptor-stimulated adenylyl cyclase activity.
  • Figure 6A the addition of TM VI peptide to membrane preparations at a concentration of 0.15 ⁇ g/wl significantly reduced isoproterenol-stimulated adenylyl cyclase activity (p ⁇ 0.05).
  • neither the peptide solubilization buffer (data not shown) nor control peptides (TM VI-Ala or TM VII of the D 2 dopamine receptor) had significant effects on isoproterenol-stimulated adenylyl cyclase activity.
  • the peptide IC5 Q values for the inhibition of dimer formation are very similar (2.14 + 0.05 ⁇ M and 3.2 ⁇ 0.04 ⁇ M, respectively) thus suggesting that receptor dimerization may be an important step in ⁇ 2AR-mediated signalling.
  • our data suggest a role for dimerization in receptor activity, one cannot exclude the possibility that the effect of the TM VI peptide is not directly due to an effect on the monomer: dimer equilibrium. Still, these results clearly show that this domain of the receptor is important in modulating ⁇ 2AR signal transduction.
  • the peptide represents a novel pharmacological tool for the study of receptor activity.
  • V2 vasopressin receptor see discussion above - this study, Figure 3
  • platelet activating factor receptor metabotropic glutamate receptor
  • substance P receptor neurokinin-2 receptor
  • C5a anaphylaxotoxin receptor glucagon receptor
  • the dopamine D] receptor D 2 receptor
  • the HT j g receptor the M 2 muscarinic receptor
  • M 3 muscarinic receptor see Hebert, T.E et al. , J. Biol. Chem. accepted. 1996, and references therein.
  • GPCR-peptides and peptidomimetic compounds could be designed for these receptors that would function to as demonstrated in these examples to selectively prevent or disrupt the functional aggregation of these receptors, thereby attenuating receptor activity.

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EP97928072A 1996-07-01 1997-06-30 Auf der messung der rezeptoroligomerisation beruhendes verfahren zum testen von verbindungen, die aktivität vom g-protein gekoppelten rezeptoren beeiflussen Withdrawn EP0939902A2 (de)

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