AU755847B2 - Polycation-sensing receptor in aquatic species and methods of use thereof - Google Patents

Description

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT

(ORIGINAL)

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4 Name of Applicant: Actual Inventor(s): Brigham and Women's Hospital William H. HARRIS, Edward BROWN and Steven

HEBERT

DAVIES COLLISON CAVE, Patent Attorneys, Level 3, 303 Coronation Drive, Milton, Queensland, 4064, Australia Address for Service: Invention Title: "Polycation-sensing receptor in aquatic species and methods of use thereof' Details of Parent Application No: 25926/97 The following statement is a full description of this invention, including the best method of performing it known to us: WO 97/35977 PCT/US97/05031 -1- POLYCATION-SENSING RECEPTOR IN AQUATIC SPECIES AND METHODS OF USE THEREOF GOVERNMENT SUPPORT This invention was made with Government support under Contract No. R01 DK38874 awarded by the National Institutes of Health. The Government has certain rights in the invention.

10 RELATED APPLICATIONS This application is a continuation-in-part of prior Serial No. 08/622,738 filed March 27, 1996, the teachings of which are hereby incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION It is well recognized that a stagnation or decline in .production of edible seafood, in particular, fish, by the marine fishing industry has occurred on a world wide basis.

20 Since the world's population increases by approximately 100 million each year, maintenance of the present caloric content of the average diet will require production of an additional 19 million metric tons of seafood per year (United Nations Food and Agriculture Organization, The State of the World Fisheries and Aquaculture, Rome, Italy (1995)). In addition, fish products are becoming increasingly utilized in ways other than just food, for example, production of shells and pearls. To achieve this level of production, aquaculture (the cultivation of marine species) will have to double its production in the next years, and wild populations of marine species must be restored.

Aquatic species includes marine teleost and elasmobranch fishes, fresh water teleost fish, euryhaline WO 97/35977 PCT/US97/05031 -2fish crustations, mousks and echinoderms. Marine teleost fish live in sea water with a high osmolality of about 1,000 mOsm. Freshwater teleost fish normally live in water of less than 50 mOsm. Euryhaline fish have the ability to acclimate to either of these environments. Ionic composition and osmolality of fish body fluids are maintained in these vastly different environments through gill, kidney and gastrointestinal tract epithelial cell function.

S 10 A major problem in aquaculture is development of methodology to rear marine teleost fish, such as cod, flounder and halibut, under freshwater hatchery conditions.

To date, factors critical to the acclimation and survival of marine species to fresh water environments, and the control of these factors, have not been fully elucidated.

~Attempts to develop such methodologies have also been complicated by problems with feeding the maturing larval forms of these fish. Development of cod, halibut or flounder species that could be reared in fresh water would be of great potential benefit in this regard. Under controlled fresh water conditions, developing forms of these fish could be raised in the absence of bacterial contamination normally present in seawater, and utilize new fresh water food sources that would potentially improve their survival.

The aquaculture industry utilizes the ability of young fish, salmon, (also called par) to be raised initially in fresh water and subsequently to be transferred for "growth out" in salt water pens as a means to produce large numbers of adult fish (young salmon tolerant to seawater are called smolt). Improvements in both the survival and health of fish undergoing the par-smolt transition would be very valuable for aquaculture growers.

WO 97/35977 PCT7US97/05031 -3- Moreover, salmon that are kept in coastal marine "grow-out" pens during the winter are constantly at risk, since both winter storms, as well as exposure to extremely cold seawater, causes fish to freeze and die. These risks are further complicated by the fact that when adult salmon are adapted to salt water they do not readily readapt back to fresh water environment. Hence, lack of understanding of the means to readapt adult salmon from salt to fresh water results in the loss of salmon.

It is apparent, therefore, that there is an immediate need to develop methods of augmenting the survival of fish in fresh water and sea water, both in a natural environment and an aquacultural environment.

SUMMARY OF THE INVENTION The present invention relates to the identification and characterization of a polyvalent cation-sensing S" receptor protein (also referred to herein as the Aquatic polyvalent cation-sensing receptor, or Aquatic PVCR) which 20 is present in various tissues of marine species. As defined herein, aquatic species includes fish (elasmobranch fish, such as sharks, skates; teleost fish, such as flounder, salmon, cod, halibut, lumpfish and trout), crustaceans lobster, crab and shrimp) and mollusks clams, mussels and oysters).

As described herein, for the first time, a polyvalent cation-sensing receptor protein has been identified in aquatic species, located on the plasma membranes of cells in the gastro-intestinal tract, kidney, ovary, lung, brain and heart, and in fish brain, gill, heart, intestines, urinary bladder, rectal gland and kidney tubules. The widespread distribution of Aquatic PVCR protein on the plasma membranes of epithelial cells, as well as in the brain, indicates the involvement of Aquatic PVCR in WO 97/35977 PCT/US97/05031 -4modulation of epithelial ion and water transport and endocrine function. Data presented herein demonstrate that the Aquatic PVCR plays a critical role in the acclimation of fish to environments of various salinities. The Aquatic polyvalent cation-sensing receptor allows the successful adaptation of fish, such as flounder, to marine and fresh water environments.

One embodiment of the present invention encompasses Aquatic PVCR proteins expressed in tissues of marine species. Aquatic PVCR proteins have been identified as being present in selected epithelial cells in marine, fresh water and euryhaline fish kidney, intestine, gill, urinary bladder, and brain. More specifically, the Aquatic PVCR protein has been identified on the plasma membranes of epithelial cells of fish kidney tubules, especially in the collecting duct (CD) and late distal tubule (LDT). The present invention is intended to encompass these Aquatic S "PVCR proteins, their amino acid sequences, and nucleic acid sequences, (DNA or RNA) that encode these Aquatic PVCR proteins.

4* In another embodiment of the present invention, methods for regulating salinity tolerance in fish are encompassed. Data presented herein indicate that the Aquatic PVCR is a "master switch" for both endocrine and kidney regulation of adult fish kidney and intestinal ion and water transport, as well as key developmental processes within the fish embryo. Modulating the expression of the Aquatic polyvalent cation-sensing receptor will activate or inhibit Aquatic PVCR mediated ion transport and endocrine changes that permit fish to adapt to fresh or salt water.

For example, methods are provided to increase the salinity tolerance of fish adapted to fresh water environment by activation of the Aquatic PVCR in selected epithelial cells. Methods are also provided to decrease WO 97/35977 PCTJUS97/05031 the salinity tolerance of fish adapted to a salt water environment by inhibiting the activity of the Aquatic PVCR in selected epithelial cells.

In another embodiment of the present invention, methods are provided to identify a substance capable of regulating ionic composition of fish fluids, salinity tolerance in fish), and endocrine function, by determining the effect that the substance has on the activation or inhibition of the Aquatic CaR. As described 10 herein, the nucleic acid sequence encoding an Aquatic PVCR eeeo has been determined and recombinant PVCR proteins can be expressed in oocytes of the frog, Xenopus laevis.

The oocyte assay system permits the screening of a large **.library of compounds that will either activate or inhibit Aquatic PVCR function. Candidate compounds can be further screened in an in vitro assay system using isolated flounder bladder preparations to measure transepithelial transport of ions important for salinity adaption.

As a result of the work described herein, Aquatic PVCR 20 proteins have been identified and their role in maintaining osmoregulation has been characterized. As a further result of the work described herein, methods are now available to modulate the activation of the Aquatic CaR, resulting in methods to regulate salinity tolerance in marine and fresh water species of fish and thus, facilitate aquaculture of marine fish. Methods of regulating salinity tolerance also provides the means to develop new species of marine fish that are easily adaptable to fresh water aquaculture.

Successful development of new species of marine fish would permit these species to be raised initially in protected fresh water hatcheries and later transferred to marine conditions.

WO 97/35977 PCT/US97/05031 -6- BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-F are photographs of immunocytochemistry results showing the distribution of PVCR protein in various tissues of elasmobranch fish, including dogfish shark (Squalus acanthias) and little skate (Raja crinacca).

Figures 2A-F are photographs of immunocytochemistry results showing the distribution of PVCR protein in various tissues of teleost fish including flounder *'..(Pseudopleuronectes americanus), trout (Onchorhychus nerka) 10 and killifish (Fundulus heteroclitus).

Figures 3A-B are audioradiograms showing RNA blotting analyses.

Figures 4A-G depict the nucleotide sequence of Shark Kidney Calcium Receptor Related Protein (SKCaR-RP) (SEQ ID S 15 NO: 1) with the ORF starting at nt 439 and ending at 3516.

Figures 5A-B depict the deduced amino acids sequence of the Shark Kidney Calcium Receptor Related Protein (SKCaR-RP) (SEQ ID NO: 2).

Figure 6 is an autoradiogram showing the results of 20 Northern blot analyses of A+ RNA from various shark tissues.

Figures 7A-B are autoradiograms showing the results of RT-PCR amplifications of poly A+RNA from various aquatic species.

Figure 8 is a photograph of immunocytochemistry results showing PVCR expression in selected tissues of Fundulus after 18 days of exposure to either sea or fresh water as determined by RNA blotting analysis.

Figures 9A-D are photographs showing the results of immunocytochemistry analysis of PVCR expression in the kidney tubules of Fundulus fish either chronically (18 days) or acutely (7 days) adapted to either salt or fresh water.

WO 97/35977 PCT/US97/05031 -7- DETAILED DESCRIPTION Described herein, for the first time, is a cell surface receptor, called the polyvalent cation-sensing receptor protein, which is present in selected epithelial cells in aquatic species tissue and organs, such as fish kidney, intestine, bladder, rectal gland, gill and brain.

This Aquatic receptor protein is also referred to herein as the Aquatic PVCR. Evidence is also presented herein that the expression of Aquatic PVCR is modulated in fish transferred from fresh to salt water. The combination of these data and knowledge of osmoregulation in fish, and other marine species, outlined briefly below, strongly suggest that Aquatic PVCR is the "master switch" for both endocrine and kidney regulation of marine species kidney, 15 intestine ion and water transport. In addition, Aquatic PVCR function may control or strongly influence maturation and developmental stages in marine species.

In mammals, calcium/polyvalent cation-sensing receptor proteins, or terrestial CaR proteins (also refereed to 20 herein as mammalian CaR, have been identified in various tissues in humans and rat. A mammalian CaR protein has been isolated and shown to be the cell surface receptor enabling mammalian parathyroid and calcitonin cells to respond to changes in extracellular Ca 2 (Brown, E.M. et al., New Eng. J. Med., 333:243, (1995)). Mammalian CaR is a membrane protein that is a member of the G-proteincoupled receptor family. When activated by external Ca 2 PVCR modulates various intracellular signal transduction pathways and alters certain functions in selected cells including secretion of various hormones (PTH, calcitonin, ACTH and prolactin) by endocrine/brain cells and ion transport by epithelial cells.

Subsequent work has revealed that abundant CaR is present in epithelial cells of the thick ascending limb /US97/05031 -8- (TAL) and distal convoluted tubules (DCT) of the mammalian kidney where it modulates transepithelial salt transport (Riccardi, D.J. et al., Proc. Nat. Acad. Sci USA, 92:131-135 (1995)). Recent research demonstrated that PVCR is present on the apical surface of epithelial cells of the mammalian kidney medullary collecting duct where it senses urinary Ca 2 and adjusts vasopressin-mediated water reabsorption by the kidney (Sands, J.M. et al., J. Clin. Invest. 99:1399- 1405 (1997)). Lastly, PVCR is also present in various regions of the brain where it is involved in regulation of thirst and associated behavior (Brown, E.M. et al., New England J. of Med., 333:234-240 (1995)).

Another protein important for osmoregulation in mammals is the NaC1 cotransporter. The NaC1 cotransporter 15 is present in the DCT of human kidney where it absorbs NaC1 and facilitates reabsorption of Ca 2 A NaC1 cotransporter protein has also been isolated from flounder urinary bladder (Gamba, G. et al., Proc. Nat. Acad. Sci. (USA), 90-2749-2753 (1993)). Recently, it has been demonstrated that NaCl reabsorption mediated by this NaC1 transporter in the DCT of humans is modulated by mammalian PVCR (Plotkin, M. et al. J.

Am. Soc. Nephrol., 6:349A (1995)).

As described herein a calcium/polycation-sensing receptor protein (referred to herein as Aquatic CaR) has 25 also been identified in specific epithelial cells in tissues critical for ionic homeostasis in marine species. It is reasonable to believe that the Aquatic PVCR plays similar critical roles in biological functions in marine species, as the mammalian PVCR in mammals.

Specifically, Aquatic PVCR proteins have been found in species of elasmobranchs and species of teleosts.

Elasmobranchs are cartilaginous fish, such as sharks, rays and skates, and are predominately marine; teleosts, such as WO 97/35977 PCT/US97/05031 -9flounder, cod, trout, killifish and salmon, can be freshwater, marine or euryhaline.

Marine teleost fish live in seawater possessing a high osmolality (1,000 mOsm) that normally contains millimolar (mM) Ca 2 50 mM Mg 2 and 450 mM NaCl (Evans, D.H. Osmotic and Ionic Regulation, Chapter 11 in The Physiology of Fishes, CRC Press, Boca Raton, FL (1993)).

Since their body fluids are 300-400 mOsm, these fish are obligated to drink sea water, absorb salts through their 10 intestine and secrete large quantities of NaCl through their gills and Mg2+ and Ca 2 through their kidneys. Their kidneys produce only small amounts of isotonic urine.

In contrast, fresh water teleost fish possess body fluids of 300 mOsm and normally live in water of less than 50 mOsm containing 5-20 mM NaCl and less than 1 mM Ca 2 and Mg 2 These fish drink little, but absorb large amounts of water from their dilute environment. As a result, their kidneys produce copious dilute urine to maintain water balance. Freshwater fish gill tissue has a low 20 permeability to ions and gill epithelial cells extract NaCl :from water (Evans, "Osmotic and Ionic Regulation", Chapter 11 in The Physiology of Fishes, CRC Press, Boca Raton, FL (1993)).

Euryhaline fish acclimate to various salinities by switching back and forth between these two basic patterns of ion and water transport. For example, when fresh water adapted teleost fish are challenged with high salinities, their gill epithelia rapidly alter net NaCl flux such that NaCl is secreted rather than reabsorbed (Zadunaisky, J.A.

et al., Bull. MDI Biol. Lab., 32:152-156 (1992)).

Reduction of extracellular Ca 2 from 10 mM to 100 pM profoundly inhibits this transport process (Zadunaisky, J.A. et al., Bull. MDI Biol. Lab., 32:152-156 (1992)). In flounder species, transfer to seawater activates a series WO 97/35977 PCT/US97/05031 of changes in the kidney allowing for secretion of large quantities of Ca2+ and Mg 2 by renal epithelia and recovery of water via a thiazide sensitive NaC1 cotransporter in the urinary bladder (Gamba, G. et al., Proc. Nat. Acad. Sci.

(USA), 90-2749-2753 (1993)).

In a similar fashion, adaption of marine euryhaline fish to fresh water is possible because of a net reversal of epithelial ionic gradients such that NaCl is actively reabsorbed and divalent metal ion secretion ceases 10 (Zadunaisky, J.A. et al., Bull. MDI Biol. Lab., 32:152-156 (1992)). These changes are mediated by alterations in hormones, especially prolactin, cortisol and arginine vasotocin (Norris, "Endocrine Regulation of lono- Osmotic Balance in Teleosts", Chapter 16 in Vertebrate Endocrinology, Lea and Febiger, Philadelphia, PA (1985)).

These alterations in a cluster of critical hormones and functional changes in epithelial transport in gill, intestine, bladder and kidney are vital not only to rapid euryhaline adaption but also throughout development of fish embryos, larvae and during metamorphosis.

As described in detail in Example i, Aquatic PVCR protein has been localized on the plasma membrane of selected epithelial cells in marine species. Specifically, Aquatic PVCR has been located on the apical membrane of epithelial cells of the collecting duct and late distal tubule of the elasmobranch kidney. Aquatic PVCR protein has also been found on the apical membranes of epithelial cells in kidney tubules, gill, urinary bladder and intestine of teleosts. As used herein, the term "apical membrane" or "apical side" refers to the "outside" of the epithelial cell exposed to urine, rather than the basal side of the cell exposed to the blood. The apical membrane is also referred to herein as facing the lumen, or interior of the kidney tubule or intestine.

WO 97/35977 PCTIUS97/05031 -11- Aquatic PVCR was also found in specific regions of teleost brain.

Aquatic PVCR protein described herein can be isolated and characterized as to its physical characteristics molecular weight, isoelectric point) using laboratory techniques common to protein purification, for example, salting out, immunoprecipation, column chromatography, high pressure liquid chromatography or electrophoresis. Aquatic PVCR proteins referred to herein as "isolated" are Aquatic PVCR proteins separated away from other proteins and cellular material of their source of origin. These isolated Aquatic PVCR proteins include essentially pure protein, proteins produced by chemical synthesis, by combinations of biological and chemical synthesis and by 15 recombinant methods.

Aquatic PVCR proteins can be further characterized as to its DNA and encoded amino acid sequences as follows: A complementary DNA (cDNA) encoding a highly conserved region of the mammalian CaR, as described in Brown, E.G. et al., 20 Nature, 366:575-580 (1993) or Riccardi, D.J. et al., Proc.

Nat. Acad. Sci USA, 92:131-135 (1995), the teachings of which are incorporated by reference, can be used as a probe to screen a cDNA library prepared from flounder urinary bladder cells to identify homologous receptor proteins. Techniques for the preparation of a cDNA library are well-known to those of skill in the art. For example, techniques such as those described in Riccardi, D.J. et al., Proc. Nat. Acad. Sci USA, 92:131-135 (1995), the teachings of which are incorporated herein by reference, can be used. Positive clones can be isolated, subcloned and their sequences determined. Using the sequences of either a full length or several partial cDNAs, the complete nucleotide sequence of the flounder PVCR can be obtained and the encoded amino acid sequence deduced. The sequences WO 97/35977 PCT/US97/05031 -12of the Aquatic PVCR can be compared to mammalian CaRs to determine differences and similarities. Similar techniques can be used to identify homologous Aquatic PVCR in other marine species.

Recombinant Aquatic PVCR proteins can be expressed according to methods well-known to those of skill in the art. For example, PVCR can be expanded in oocytes of the frog, Xenopus laevis, both to prove identity of the cDNA clone and to determine the profile of activation of Aquatic 10 PVCR proteins as compared to mammalian CaR proteins.

S. Exemplary techniques are described in (Brown, E.G. et al., S. Nature, 366:575-580 (1993); Riccardi, D.J. et al., Proc.

Nat. Acad. Sci USA, 92:131-135 (1995)), the teachings of which are incorporated herein by reference.

15 As described in Example 2, a 4.4 kb homolog of the mammalian CaR has been found in flounder urinary bladder together with abundant 3.8 kb thiazide-sensitive NaCl cotransporter transcript. Using a homology cloning strategy, a cDNA library from dogfish shark kidney was prepared and screened to obtain multiple cDNA clones with partial homology to mammalian CaRs as described in Example 3. One clone called Shark Kidney Calcium Receptor Related Protein (SKCaR-RP) was isolated and characterized. SKCaR- RP (also referred to herein as Shark Aquatic PVCR) is 4,131 nucleotides in size (SEQ ID NO: As shown in Figure 4, the complete nucleotide sequence of SKCaR-RP reveals that the clone is composed of 438 nts of 5' untranslated region or UTR followed by a single open reading frame (ORF) of 3,082 nts followed by 610 nts of 3' UTR containing regions of poly A+ RNA.

Figure 5 shows the ORF of the SKCaR-RP in single letter amino acid designations (SEQ ID NO: The deduced amino acid sequence of SKCaR-RP predicts a protein of approximately 110,00 daltons that is 74% homologous to both WO 97/35977 PCT/US97/05031 -13the rat kidney PVCR protein as well as bovine parathyroid PVCR protein. Analysis of the amino acid sequence reveals that SKCaR-RP possesses general features that are homologous to PVCR proteins including a large extracellular domain, 7 transmembrane domains and cytoplasmic carboxyl terminal domain. In this regard, many amino acids demonstrated to be critical to PVCR function are identical in SKCaR-RP as compared to mammalian PVCR proteins including specific regions of the extracellular domain and 10 the 7 transmembrane domains. In contrast, other regions are highly divergent, including the amino acids number 351-395 in the extracellular domain as well as the most of the carboxyl terminal region amino acids 870- 1027).

Importantly, the region of amino acids present in mammalian 15 CaRs that was used to generate anti-CaR antiserum is also present in SKCaR-RP.

As shown in Figure 6, Northern blot analysis of mRNA from various shark tissues reveals the highest degree of SKCaR-RP in gill followed by kidney and then rectal gland.

i 20 These data are highly significant since these tissues have been demonstrated to be involved with ion and water transport and body homeostasis and possess epithelial cells that stain with anti-CaR antiserum. There appears to be at least 3 distinct mRNA species of approximately 7 kb, 4.2 kb and 2.6 kb that hybridize to SKCaR-RP. The 4.2 kb likely corresponds to the SKCaR-RP clone described above.

RT-PCR amplications were performed as described in SExample 3 after isolation of poly A+ RNA from various aquatic species. Primers that permit selective amplification of a region of CaRs (nts 597-981 of RaKCaR ScDNA) that is 100% conserved in all mammalian CaRs were utilized to obtain the sequences of similar CaRs in aquatic species. These primers amplify a sequence of 384 nt that is present in the extracellular domain of CaRs and WO 97/35977 PCTIUS97/05031 -14presumably is involved in binding divalent metal ions. The resulting amplified 384 bp cDNA was ligated into a cloning vector and transformed into E. coli cells for growth, purification and sequencing.

As shown in Figures 7A and B, partial cDNA clones have been obtained from: dogfish shark kidney (lane flounder urinary bladder (lane lumpfish liver (lane lobster muscle (lane clam gill (lane 9) and sea cucumber 0' respiratory tissue (lane 10) using these identical 10 primers. Some tissues (flounder brain-lane 7) did not yield a corresponding 384 nt cDNA despite careful controls.

Similarly, no 384 nt cDNA was obtained when only water and not RT reaction mixture was added. These data suggest these 384 nt cDNAs are specific and not expressed in all S 15 tissues of aquatic organisms. Each of these 384 nt cDNAs was sequenced and found to contain a conserved nucleotide sequence identical to that present in mammalian CaRs.

These data suggest the presence of CaR related proteins in classes of aquatic organisms that are widely divergent in evolution. These include teleost fish (flounder, lumpfish), elasmobranch fish (dogfish shark), crustaceans (lobster), mollusks (clam) and echinoderms (sea cucumber).

It is important to note that Aquatic PVCR sequence obtained from these clones shared complete identity of the 384 nt segment of mammalian CaRs. However, the Aquatic PVCR sequence obtained from the shark kidney clone did not.

These data suggest that at least two different classes of aquatic polyvalent cation-sensing receptors exist.

The present invention is intended to encompass Aquatic PVCR proteins, and proteins and polypeptides having amino acid sequences analogous to the amino acid sequences of Aquatic PVCR proteins. Such polypeptides are defined herein as Aquatic PVCR analogs homologues), or derivatives. Analogous amino acid sequences are defined WO 97/35977 PCTIUS97/05031 herein to mean amino acid sequences with sufficient identity of Aquatic PVCR amino acid sequence to possess the biological activity of an Aquatic PVCR. For example, an analog polypeptide can be produced with "silent" changes in the amino acid sequence wherein one, or more, amino acid residues differ from the amino acid residues of the Aquatic PVCR protein, yet still possesses the biological activity of Aquatic PVCR. Examples of such differences include additions, deletions or substitutions of residues of the 10 amino acid sequence of Aquatic PVCR. Also encompassed by the present invention are analogous polypeptides that exhibit greater, or lesser, biological activity of the Aquatic PVCR proteins of the present invention.

The "biological activity" of Aquatic PVCR proteins is defined herein to mean the osmoregulatory activity of Aquatic PVCR mammalian PVCR proteins have been shown to 0:0"0: mediate physiological responses to changes in body osmolality and salt content in kidney, parathyroid, calcitonin and brain cells. (Brown, E.M. et al., New Enq.

.0 20 J. Med., 333:243, (1995); Riccardi, D.J. et al., Proc. Nat.

Acad. Sci USA, 92:131-135 (1995); Sands, J.M. et al., Nature (Medicine) (1995); Brown, E.M. et al., New England J. of Med., 333:234-240 (1995)). It is reasonable to believe that Aquatic PVCR proteins will possess identical, or similar osmoregulatory activities as these previously identified mammalian CaR proteins in fish kidney, gill, bladder, intestine, rectal gland and brain cells. Assay techniques to evaluate the biological activity of Aquatic PVCR proteins and their analogs are described in Brown, E.M. et al., New Enq. J. Med., 333:243, (1995); Riccardi, D.J. et al., Proc. Nat. Acad. Sci USA, 92:131-135 (1995); Sands, J.M. et al., Nature (Medicine) (1995); Brown, E.M.

et al., New England J. of Med., 333:234-240 (1995), the teachings of which are incorporated herein by reference.

WO 97/35977 PCT/US97/05031 -16- Additional assays to evaluate biological activity of PVCR proteins are described in U.S. Serial No. 60/003,697, the teachings of which are also incorporated herein, in its entirety, by reference.

The "biological activity" of Aquatic PVCR is also defined herein to mean the ability of the Aquatic PVCR to modulate signal transduction pathways in specific marine species cells. In mammals, studies in normal tissues, in oocytes using recombinantly expressed CaR, and cultured 10 cells have demonstrated that mammalian CaR protein is capable of complexing with at least two distinct types of GTP-binding proteins that transmit the activation of CaR by an increase in extracellular calcium to various intracellular signal transduction pathways. One pathway consists of mammalian CaR coupling with an inhibitory Gi protein that, in turn, couples with adenylate cyclase to reduce intracellular cAMP concentrations. A second distinct pathway consists of CaR coupling to stimulatory Gq/Gall G protein that couples with phospholipase C to generate inositol 1,4,5 triphosphosphate that, in turn, stimulates both protein kinase C activity and increases intracellular Ca2+ concentrations. Thus, depending on the distribution and nature of various signal transduction pathway proteins that are expressed in cells, biologically active mammalian CaRs modulate cellular functions in either an inhibitory or stimulatory manner. It is reasonable to believe that biologically active Aquatic PVCR possesses similar signal transduction activity.

The present invention also encompasses biologically active polypeptide fragments of the Aquatic PVCR proteins described herein. Such fragments can include only a part of the full-length amino acid sequence of an Aquatic PVCR yet possess osmoregulatory activity. For example, polypeptide fragments comprising deletion mutants of the WO 97/35977 PCT/US97/05031 -17- Aquatic PVCR proteins can be designed and expressed by well-known laboratory methods. Such polypeptide fragments can be evaluated for biological activity as described herein.

Antibodies can be raised to the Aquatic PVCR proteins and analogs, using techniques known to those of skill in the art. These antibodies polyclonal, monoclonal, chimeric, or fragments thereof, can be used to immunoaffinity purify or identify Aquatic PVCR proteins 10 contained in a mixture of proteins, using techniques wellknown to those of skill in the art. These antibodies, or antibody fragments, can also be used to detect the presence of Aquatic PVCR proteins and homologs in other tissues using standard immunochemistry methods.

15 The present invention also encompasses isolated nucleic acid sequences encoding the Aquatic PVCR proteins described herein, and fragments of nucleic acid sequences encoding biologically active PVCR proteins. Fragments of the nucleic acid sequences described herein as useful as probes to detect the presence of marine species CaR.

Specifically provided for in the present invention are DNA/RNA sequences encoding Aquatic PVCR proteins, the fully complementary strands of these sequences, and allelic variations thereof. Also encompassed by the present invention are nucleic acid sequences, DNA or RNA, which are substantially complementary to the DNA sequences encoding Aquatic PVCR, and which specifically hybridize with the Aquatic PVCR DNA sequences under conditions of stringency known to those of skill in the art, those conditions being sufficient to identify DNA sequences with substantial nucleic acid identity. As defined herein, substantially complementary means that the sequence need not reflect the exact sequence of Aquatic PVCR DNA, but must be sufficiently similar in identity of sequence to hybridize -18with Aquatic PVCR DNA under stringent conditions.

Conditions of stringency are described in Ausebel, et al., Current Protocols in Molecular Biology, (Current Protocols, 1994). For example, non-complementary bases can be interspersed in the sequence, or the sequences can be longer or shorter than Aquatic PVCR DNA, provided that the sequence has a sufficient number of bases complementary to Aquatic PVCR to hybridize therewith.

Exemplary hybridization conditions are described herein and 10 in Brown, et al. Nature, 366:575 (1993). For example, conditions such as IX SSC 0.1% SDS, 50 0 C, or SSC, 0.1% SDS, 50 0 C can be used as described in Examples 2 and 3.

15 For example, a representative nucleic acid that is substantially complementary to SEQ ID NO:1 is set forth in SEQ ID NO:3. This nucleic acid is 99% identical to SEQ ID NO:1 and encodes a polypeptide (SEQ ID NO:4) that is 97% identical to the polypeptide set forth in •SEQ ID NO:2.

e S. 20 The Aquatic PVCR DNA sequence, or a fragment thereof, can be used as a probe to isolate additional Aquatic PVCR homologs. For example, a cDNA or genomic DNA library from the appropriate organism can be screened with labeled Aquatic PVCR DNA to identify homologous genes as described in Ausebel, et al., Current Protocols in Molecular Biology, (Current Protocols, 1994).

Typically the nucleic acid probe comprises a nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO:3, or fragments thereof) and is of sufficient length and complementarity to specifically hybridize to nucleic acid sequences which encode Aquatic species PVCR. The requirements of sufficient length and complementarity can be easily determined by one of skill in the art.

18A- As described in Example 4, it is demonstrated that the Aquatic PVCR protein plays a critical role in the adaption of euryhaline fish to environments of various salinities.

Adaption of the killifish, Fundulus heteroculitus, to seawater resulted in steady state expression of Aquatic PVCR mRNA in various tissues.

It is also demonstrated herein that PVCR protein undergoes rearrangement within epithelial cells of the e* WO 97/35977 PCT/US97/05031 -19urinary bladder in flounder adapted to brackish water as compared to full strength sea water. This directly correlates with alterations the rate of NaCl transport by these cells.

Winter flounder were adapted to live in 1/10th seawater (100 mOsm/kg) by reduction in salinity from 450 mM NaCl to 45 mM NaC1 over an interval of 8 hrs. After a day interval where these fish were fed a normal diet, the distribution of the PVCR in their urinary bladder 10 epithelial cells was examined using immunocytochemistry.

PVCR immunostaining is reduced and localized primarily to the apical membrane of epithelial cells in the urinary bladder. In contrast, the distribution of PVCR in .epithelial cells lining the urinary bladders of control flounders continuously exposed to full strength seawater is more abundant and present in both the apical membranes as well as in punctate regions throughout the cell. These data are consistent with previous Northern data since more PVCR protein is present in the urinary bladders of seawater 20 fish vs fish adapted to brackish water. These data suggest that PVCR protein may be present in vesicles in epithelial cells of the urinary bladder and that in response to alterations in salinity, these vesicles move from the cell cytoplasm to the apical surface of these epithelial cells.

Since these same epithelial cells possess abundant NaCl cotransporter protein that is responsible for water reabsorption in the urinary bladder, these data suggest that the PVCR protein modulates NaCl transport in the flounder urinary bladder by altering the proportion of NaCl cotransporter protein that is present in the apical membrane. As urinary Mg 2 and Ca2+ concentrations increase when fish are present in full strength sea water, activation of apical PVCR protein causes endocytosis and WO 97/35977 PCTUS97/05031 removal of NaCl cotransporter from the apical membrane and thus reduction in urinary bladder water transport.

As a result of the work described herein, methods are now provided that facilitate euryhaline adaptation of fish to occur, and improve the adaption. More specifically, methods are now available to regulate salinity tolerance in fish by modulating (or alternating) the activity of the Aquatic PVCR protein present in epithelial cells involved in ion transport, as well as in endocrine and nervous 10 tissue. For example, salinity tolerance of fish adapted (or acclimated) to fresh water can be increased by activating the Aquatic PVCR, for example, by increasing the expression of Aquatic PVCR in selected epithelial cells, resulting in the secretion of ions and seawater adaption.

Specifically, this would involve regulatory events controlling the conversion of epithelial cells of the gill, intestine and kidney. In the kidney, PVCR activation will o* facilitate excretion of divalent metal ions including Ca 2 and Mg 2 by renal tubules. In the gill, PVCR activation 20 will reduce reabsorption of ions by gill cells that occurs in fresh water and promote the net excretion of ions by gill epithelia that occurs in salt water. In the intestine, PVCR activation will permit reabsorption of water and ions across the G.I. tract after their ingestion by fish.

Alternatively, the salinity tolerance of fish adapted to seawater can be decreased by inhibiting the Aquatic PVCR, for example, by decreasing the expression of Aquatic PVCR in selected epithelial cells, resulting in alterations in the absorption of ions and freshwater adaption.

Selected epithelial cells include, kidney, bladder, intestinal and gill cells.

The presence of Aquatic PVCR in brain reflects both its involvement in basic neurotransmitter release via WO 97/35977 PCTIUS97/05031 -21synaptic vesicles (Brown, E.M. et al., New England J. of Med., 333:234-240 (1995)), as well as its activity to trigger various hormonal and behavioral changes that are necessary for adaptation to either fresh water or marine environments. For example, increases in water ingestion by fish upon exposure to salt water is mediated by PVCR activation in a manner similar to that described for humans :where PVCR activation by hypercalcemia in the subfornical organ of the brain cause an increase in water drinking 1 0 behavior (Brown, E.M. et al., New England J. of Med., 333:234-240 (1995)). In fish, processes involving both alterations in serum hormonal levels and behavioral changes are mediated by the brain. These include the reproductive *and spawning of euryhaline fish in fresh water after their migration from salt water as well as detection of salinity of their environment for purposes of feeding, nesting, migration and spawning.

Data obtained recently from mammals now suggest that PVCR activation may play a pivotal role in coordinating 20 these events. For example, alterations in plasma cortisol have been demonstrated to be critical for changes in ion transport necessary for adaptation of salmon smolts from fresh water to salt water (Veillette, et al., Gen.

and Comp. Physiol., 97:250-258 (1995). As demonstrated recently in humans, plasma Adrenocorticotrophic Hormone (ACTH) levels that regulate plasma cortisol levels are altered by PVCR activation.

The term "activation" as used herein means to make biologically functional, rendering a cell surface receptor capable of stimulating a second messenger which results in modulation of ion secretion. This could be in the form of either an inhibition of signal transduction pathways, via a Gi protein, or stimulation of other pathways via. a Gq/Gall protein. As a result of WO 97/35977 PCT/US97/05031 -22these alterations, ion transport by epithelial cells is reduced or stimulated.

For example, a compound, or substance, which acts as an agonist can interact with, or bind to, the Aquatic CaR, thereby activating the Aquatic CaR, resulting in an increase of ionsecretion in selected epithelial cells. An agonist can be any substance, or compound, that interacts with, or binds to, the Aquatic PVCR resulting in activation of Aquatic CaR. Agonists encompassed by the present 10 invention include inorganic ions, such as the polyvalent cations calcium, magnesium and gadolinium, and organic molecules such as neomycin. Other agonists, include inorganic compounds, nucleic acids or proteins can be determined using the techniques described herein.

Agonists also encompassed by the present invention can include proteins or peptides or antibodies that bind to the Aquatic PVCR resulting in its activation. Activation of the Aquatic PVCR is typically direct activation. For example, an inorganic molecule or peptide binds directly to 20 the receptor protein resulting in the activation of Aquatic CaR. However, activation of the Aquatic PVCR can also be indirect activation, such as would occur when an antibody is available to bind an Aquatic PVCR antagonist, thus permitting activation of the Aquatic PVCR The term "deactivation" or "inactivation" as used herein means to completely inhibit or decrease biological function. For example, deactivation is when a cell surface receptor is incapable of stimulating a second messenger.

Specifically, as used herein, deactivation of the Aquatic PVCR occurs when the Aquatic PVCR is rendered incapable of coupling with, or stimulating, a second messenger, resulting in the absorption of ions in selected epithelial cells. Deactivation can be direct or indirect. For example, an antagonist can interact with, or bind directly WO 97/35977 PCT/US97/05031 -23to the Aquatic PVCR, thereby rendering the Aquatic PVCR incapable of stimulation of a messenger protein.

Alternatively, deactivation can be indirect. For example, an antagonist can deactivate Aquatic PVCR by preventing, or inhibiting an agonist from interacting with the Aquatic CaR. For example, a chelator can bind calcium ions and, thus prevent the calcium ions from binding to the Aquatic

PVCR.

Antagonists of the Aquatic PVCR can be any substance 10 capable of directly interacting with, or binding to, the Aquatic PVCR or interacting with, or binding to, an agonist of the Aquatic PVCR that results in deactivation of the Aquatic PVCR. Antagonists encompassed by the present invention can include, for example, inorganic molecules, organic molecules, proteins or peptides. Antagonists can also be nucleic acids, such as anti-sense DNA or RNA sequences that bind to the DNA encoding the Aquatic PVCR, thereby preventing or inhibiting transcription into mRNA.

Antagonists can also be anti-sense RNA that binds to the 20 PVCR transcript, thereby preventing, or inhibiting translation.

Candidate substances, compounds, peptides or nucleic acids) to be evaluated for their ability to regulate Aquatic PVCR activity can be screened in assay systems to determine activity. For example, one assay system that can be used is the frog oocyte system expressing Aquatic PVCR described in Brown, E.G. et al., Nature, 366:575-580 (1993); Riccardi, D.J. et al., Proc.

Nat. Acad. Sci USA, 92:131-135 (1995).

A functional assay to screen for compounds that alter PVCR mediated NaCl transport function in adult flounder urinary bladder can also be used to screen candidate compounds for their ability to modulate Aquatic PVCR.

Transport of NaCl via the thiazide sensitive NaCI WO 97/35977 PCT/US97/05031 -24cotransporter in the flounder urinary bladder is important in its adaptation to various salinities. NaCl transport is readily quantified using a isolated bladder preparation from adult flounder and measurement of transepithelial Ca 2 sensitive short circuit current, as described in (Gamba, G.

et al., Proc. Nat. Acad. Sci. (USA), 90-2749-2753 (1993)).

Use of this isolated in vitro assay system can establish a direct effect of Aquatic PVCR function or transepithelial transport of ions important for salinity adaptation.

10 Compounds identified using the frog oocyte assay and in vitro NaCl transport assay system can be further tested in whole animal adaptation experiments.

For example, to screen for PVCR reactive compounds (both agonists and antagonists) an assay previously used 15 for study of ion and water transport in isolated flounder urinary bladders (Renfro, L.J. Am. J. Physiol. 228:52-61, 1975) has been used. As described herein (Example this assay has now been adapted to screen PVCR agonists and provided data showing that water reabsorption is 20 inhibited by application of thiazide (specific inhibitor of the thiazide sensitive NaCl cotransporter); water reabsorption is >90% inhibited by application of gadolinium (a PVCR specific agonist); water reabsorption is inhibited by application of neomycin (a PVCR specific agonist); and exposure of the bladder to PVCR agonists is reversible upon removal of either gadolinium or neomycin.

As a further result of the work, methods are provided to test the function of PVCR in developing fish, and to specifically select for fish with altered PVCR functional and osmotic tolerance. The developmental expression of PVCR in developing embryo, larval and metamorphic forms of fish can be determined using antibodies that recognize Aquatic PVCR and/or mammalian CaR, or by using Aquatic and/or mammalian cDNA probes, or a combination of these WO 97/35977 PCTIUS97/05031 techniques. Initial screening of gametes, larval or metamorphic forms of fish can be tested using immunohistochemistry, such as described in Example 1, to determine at what stage of development the PVCR protein is expressed in developing fish.

Based on the immunochemistry studies of the Aquatic PVCR structure, function and developmental expression, specific selection assays can be designed to identify fish, flounder, halibut or cod, species with altered 10 Aquatic PVCR function that can survive in fresh water, while those possessing normal PVCR function will die.

These acute survival assays can evaluate the overall effect of PVCR agonists and antagonists identified by the frog oocyte expression assay. These assays will test the potency of various PVCR active compounds on improving or reducing survival of various fish or embryos. The ability to identify a single individual fish with alterations in PVCR function and osmoregulation from many wild type fish possessing normal characteristics will permit the S. 20 propagation of specific strains of fish that exhibit specific salinity tolerance characteristics. Development of larval forms of cod, halibut or flounder that survive in fresh water can then be utilized in experiments to test whether new food sources could be used in their rearing.

Successful development of these goals would then permit these species to be raised initially in protected fresh water hatcheries and later transferred to marine conditions similar to those presently utilized for aquaculture of salmon.

Also encompassed by the present invention are methods of modulating the activation of the Aquatic PVCR by altering the DNA encoding the Aquatic PVCR, and thus, altering the subsequent expression of Aquatic PVCR protein in various tissues. For example, anti-sense nucleic acid WO 97/35977 PCT/US97/05031 -26sequences (either DNA or RNA) can be introduced into e.g., epithelial cells in fish kidney, where the anti-sense sequence-binds to the Aquatic PVCR gene and inhibits, or substantially decreases its transcription into mRNA.

Alternatively, the anti-sense sequence can bind to the Aquatic PVCR mRNA and inhibit, or substantially decrease, its translation into amino acid sequence.

Alternatively, a mutated or chimeric Aquatic PVCR gene construct a mutated or chimeric SEQ ID NO: 1) can be 10 inserted into, e.g. fish eggs, to produce new marine strains with enhanced, or decreased, Aquatic PVCR protein activity. The anti-sense sequence or gene construct is introduced into the cells using techniques well-known to those of skill in the art. Such techniques are described in Hew, et al., Mol. Aquatic Biol. Biotech., 1:3807- 17 (1992) and Du, et al., Biotechnoloqy, 10:176-181 (1992), the teachings of which are incorporated herein by reference.

Based on the work described herein, new methodologies 20 that will regulate the adaptation of fish, particularly flounder, halibut and cod, to environments of varying salinities are now available. For example, methods are now available to adapt developing forms of flounder, halibut or cod to fresh water environments. Rearing of these species in fresh water will allow for new approaches to the problems of feeding and successful rearing of larval forms of these fish species. Methods are also now available for selection and propagation of new strains of fish flounder, halibut and cod) that will possess alterations in their salinity tolerance such that they can be raised in fresh water, then transferred to seawater. This approach has many advantages since it will both diversify the aquaculture industry and make use of existing hatcheries WO 97/35977 PCT/US97/05031 -27and facilities to produce flounder, cod or halibut as well as salmon.

The present invention is illustrated by the following Examples, which are not intended to be limited in any way.

EXAMPLE 1: IMMUNOHISTOCHEMISTRY OF THE PVCR PROTEIN PRESENT IN AQUATIC SPECIES EPITHELIAL

CELLS

Tissues from fish were fixed by perfusion with 2% paraformaldehyde in appropriate Ringers solution 10 corresponding to the osmolality of the fish after anesthesitizing the animal with MS-222. Samples of tissues were then obtained by dissection, fixed by immersion in 2% paraformaldehyde, washing in Ringers then frozen in an embedding compound, O.C.T.TM Miles, Inc. Elkahart, Indiana, using methylbutane cooled with liquid nitrogen.

After cutting 4pM tissue sections with a cryostat, individual sections were subjected to various staining protocols. Briefly, sections mounted on glass slides were: 1) blocked with serum obtained from the species of fish, 2) 20 incubated with rabbit anti-CaR antiserum and 3) washed and incubated with peroxidase conjugated affinity purified goat antirabbit antiserum. The locations of the bound peroxidase conjugated goat antirabbit antiserum was visualized by development of a rose colored aminoethylcarbazole reaction product. Individual sections were mounted, viewed and photographed by standard light microscopy techniques. The anti-CaR antiserum used to detect fish PVCR protein was raised in rabbits using a 23 mer peptide corresponding to amino acids numbers 214-237 localized in the extracellular domain of the RaKCaR protein.

In both species of elasmobranchs studied, (dogfish shark, Squatus Acanthias and little skate, Raja Erinacea), PVCR protein was localized to the apical membranes of -28selected epithelial cells. The distribution of PVCR in elasmobranch tissue is shown in Figures 1A-F. Heavy black coloring is displayed where anti-CaR antibody binding is present consistently in areas of tissues designated by arrowheads. Figure 1A: Kidney-CaR expression is present on apical membranes of epithelial cells of late distal tubule and collecting duct Figure 1B: Gill PVCR expression is localized to epithelial cells of gill arcades.

Figure 1C: Brain PVCR expression is localized to distinct groups of neurons in the brain. Figure ID: Rectal gland PVCR expression is localized to apical membranes of cells lining the ducts of the rectal gland. Figure lE: Intestine S, PVCR expression is localized to the apical membranes of o. epithelial cells lining the lumens of the intestine. Figure 15 1 F: Ovary PVCR expression is present in both oocytes and surrounding follicular cells.

Figures 2A-F show the distribution of PVCR in the flounder (Pseudopleuronectes americanus) and in the fresh water trout (Onchorhynchus Nerka). Figures 2A-F display 20 heavy black coloring where anti-CaR antibody binding is present consistently in areas of tissues designated by arrowheads. Figure 2A: Kidney-CaR expression is present on apical membranes of epithelial cells of large tubules (LT) and collecting ducts Figure 2B: Gill PVCR expression is localized to epithelial cells of gill arcades. Figure 2C: Brain PVCR expression is localized to distinct groups of neurons in the brain. Figure 2D: Urinary bladder PVCR expression is localized to apical membranes of cells lining the urinary bladder. Figure 2E: Intestine PVCR expression is localized to the apical membranes of epithelial cells lining the lumens of the intestine. Figure 2F: Ovary PVCR expression is present in both oocytes and surrounding follicular cells.

WO 97/35977 PCT/US97/05031 -29- EXAMPLE 2: RNA BLOTTING ANALYSES OF WINTER FLOUNDER TISSUE Five microgram samples of poly A+ RNA prepared from various winter flounder tissues including muscle (lane 1), heart (lane testis (lane 3) and urinary bladder (lane 4) were subjected to RNA blotting analyses (Figures 3A and

B).

As shown in Figure 3A, a single filter was first hybridized using a 32 P-labeled ECO R1/XHO 1 5' fragment of ::rat kidney PVCR cDNA (Brown, et al., Nature, 366:575 1 0 (1993)), washed at reduced stringency (lX SSC, 0.1% SDS, 50 0 and exposed for 10 days to autoradiography.

As shown in Figure 3B, the same filter shown in Figure after stripping and hybridization with a 32-labeled full length 3.8 kb TSC cDNA that was washed at 0.1% SDS at 650 C. and subjected to a 1 hour autoradiogram exposure. Data shown representative of a total of five separate experiments.

These data demonstrate the presence of a 4.4 kb homolog of the mammalian CaR present in poly A+ RNA from 20 urinary bladder together with abundant 3.8 kb thiazidesensitive NaC1 contransporter transcript, and suggest no PVCR transcripts are present in other tissues including muscle, heart or testis.

EXAMPLE 3: MOLECULAR CLONING OF SHARK KIDNEY CALCIUM RECEPTOR RELATED PROTEIN (SKCaR-RP) A shark XZAP cDNA library was manufactured using standard commercially available reagents with cDNA synthesized from poly A+ RNA isolated from shark kidney tissue as described and published in Siner et. al Am. J.

Physiol. 270:C372-C381, 1996. The shark cDNA library was plated and resulting phage plaques screened using a 32p labeled full length rat kidney CaR (RaKCaR) cDNA probe under intermediate stringency conditions (0.5X SSC, 0.1% WO 97/35977 PCT/US97/05031 SDS, 50 0 Individual positive plaques were identified by autoradiography, isolated and rescued using phagemid infections to transfer cDNA to KS Bluescript vector. The complete nucleotide sequence, Figure 4, (SEQ ID NO: 1) of the 4.1 kb shark kidney PVCR related protein (SKCaR-RP) clone was obtained using commercially available automated sequencing service that performs nucleotide sequencing using the dideoxy chain termination technique. The deduced amino acid sequence (SEQ ID NO: 2) is shown in Figure 10 Northern analyses were performed as described in Siner et. al. Am. J. Physiol. 270:C372-C381, 1996. The SKCaR-RP nucleotide sequence was compared to others CaRs using commercially available nucleotide and protein database services including GENBANK and SWISS PIR.

Polymerase chain reaction (PCR) amplification of selected cDNA sequences synthesized by reverse transcriptase (RT) were performed using a commercially available RT-PCR kit from Promega Biotech, Madison, WI.

Selective amplification of a conserved region of CaRs (nts 20 597-981 of RaKCaR cDNA) results in 384 nt cDNA, as shown in Figure 7. This amplified 384 bp was then ligated into the TA cloning vector (Promega Biotech, Madison, WI) that was then transformed into competent DH5a E. coli cells using standard techniques. After purification of plasmid DNA using standard techniques the 384 nt cDNA was sequenced as described above.

EXAMPLE 4: PVCR EXPRESSION IN TISSUES OF FUNDULUS

HETEROCLITUS

To determine if PVCR expression was modulated by adaptation of Fundulus to either fresh or salt water, killifish collected in an estuary were first fresh or salt water adapted for an interval of 18 days (chronic adaptation). Selected individuals from each group were then WO 97/35977 PCT/US97/05031 -31adapted to the corresponding salinity (fresh to salt; salt to fresh) for an interval of 7 days (acute adaptation).

Results are shown in Figure 8. A blot containing RNA ug/lane) prepared from control Xenopus kidney (lane 1) or Fundulus heart (containing ultimobranchial tissue) (lanes 2, kidney (lanes 3, 6) and gill (lanes 4, 7) was probed with a 32 p-labeled Xenopus PVCR cDNA, washed (.01 x SSC, 65 0 C) and autoradiographed. As shown in Figure 8, as compared to control mRNA, (lane 1) steady state levels of 10 PVCR mRNA are larger in tissues from seawater adapted fish (lanes 5-7) versus those in fresh water (lanes 2-4) Fundulus fish were either chronically (Figures 9A and 9B) or acutely (Figures 9C and 9D) adapted to salt water (Figures 9A and 9C) or fresh water (Figures 9B and 9D).

The presence of PVCR in kidney tubules was determined by immunocytochemistry. Chronic adaptation to salt water (9A) resulted in increased PVCR expression in kidney tubules as compared to that present in fresh Kidney tubule PVCR expression in salt water fish was diminished by acute S. 20 adaptation to fresh water In contrast, kidney tubule PVCR expression in fresh water fish was increased after acute adaptation to salt water (9D).

EXAMPLE 5: ASSAY FOR PVCR AGONISTS AND ANTAGONISTS USING THE FLOUNDER URINARY BLADDER To provide further evidence linking Aquatic PVCRs to fish osmoregulation, isolated urinary bladder of winter founder was used to investigate whether PVCRs modulate epithelial cell ion transport. Previous work has demonstrated that the flounder urinary bladder is important in osmoregulation since it allows recovery of both NaCl and water via a thiazide-sensitive NaC1 contransport process that has been first generated by the kidney proximal tubule. Water reabsorption from the urine stored in WO 97/35977 PCT/US97/05031 -32urinary bladder allows for the concentrations of both Mg 2 and Ca 2 to increase to values as high as 84 mM and 7 mM respectively in marine founders (Elger, et al., J.

Comp. Physiol., B157:21 (1987)).

Net apical to basolateral water flux (Jv) was measured gravimetrically in 10 minute intervals using individual urinary bladder excised from winter flounder. Briefly, isolated bladders were suspended in a liquid solution (typically a physiologically compatible solution) as 10 described in (Renfro, L.J. Am. J. Physiol. 228:52-61, 1975) the teachings of which are hereby incorporated by reference. The weight of the bladder was measured before and after the experimental .period, wherein the experimental period comprised the period of time that the isolated 15 bladder was exposed to test compound. The compound to be tested test compound) was added to both serosal and mucosal solutions. The bladders were dried and weighted as o described in Renfro et al. The difference in bladder weight prior to and after exposure to test compound is an indication of water reabsorption by the bladder.

Quantification of water reabsorption (Jv) by isolated bladders using the method of Renfro et al. showed that Jv was significantly (p<0.05) inhibited by addition of 100 jM hydrochlorothiazide consistent with the role of the thiazide sensitive NaCl contransporter in this process.

Urinary bladder Jv was also significantly inhibited by PVCR agonists including 100 AM Gd 3 and 200 pM neomycin (Control Jv values (130±28 pl/gm/hr.) were obtained from animals in September-October and are approximately 21% of the Jv reported by Renfro et al.

These differences likely reflect seasonal variations in urinary bladder transport.) The half maximal inhibitory concentration for urinary bladder Jv (IC 50 for Gd 3 AM) was similar to that reported for mammalian CaRs, while -33the IC 50 for neomycin (150 pM) was approximately 3 times larger as compared to mammalian CaRs (50 yM). This inhibitory effect of PVCR agonists on Jv was fully reversible. Activation of apical PVCRS by high concentrations of MG2+ and Ca 2 resulting from NaClmediated water reabsorption from bladder urine would :.provide for optimal recovery of water by the urinary bladder. This mechanism would permit water reabsorption t( proceed until divalent cation concentrations approach 10 levels that promote crystal formation. This overall process is similar to that described for mammalian CaRs in the rat and human IMCD. Additional aspects of these mammalian and teleost renal epithelia may also share other similarities since teleost urinary bladder is both an anatomical and functional homolog of the mammalian mesonephric kidney.

EQUIVALENTS

Those skilled in the art will recognize, or be able to 20 ascertain using no more than routine experimentation, many equivalents to specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The reference to any prior art in this specification is not, and should not be taken as, anr acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

EDITORIAL NOTE NO: 66689/00 Sequence listing pages i-xvii is part of the description.

The claims are to follow.

SEQUENCE LISTING <110> Brigham and Women's Hospital <120> Polycation-Sensing Receptor in Aquatic Species and Methods of Use Thereof <130> squalus acanthias <140> PCT/US97/05031 <141> 1997-03-27 <150> US 08/622,738 <151> 1996-03-27 <160> 4 <170> Patentln Ver. <210> 1 <211> 4131 <212> DNA <213> Squalus acanthias <220> <221> CDS <222> (439) (3519) <400> 1 aattccgttg ctgtcggttc agtccaagtc tcctccagtg caaaatgaga aatggtggtc C.

C.

S.

C.

S C C

S.

C C

C

S. S C S gccattacag tattaaaatg gtcattgtat gttcaccctt atggggattg gcagaaatcc cagagacagg gaacatgcac tacatctgtg tttctgcaag gatggcttca gaataactga ccaaagggat tcttggagca tacgatcaac atcttccagg agttctgctg tccaggcatc ctctgtaaac gctgcaca atg gct cag c Met Ala Gln I ttaatgaaat cgagaaatca gtaacaaaat cctgaaggag taaagcgatc gggctggcgt :tt cac tgc ~eu His Cys at gtc tca isn Val Ser 20 tc ata ctg le Ile Leu attgtcagtt attctgcacg ggaacaaagc at ggaagact cctcaccatt agtgtggctt caa ctc tta Gln Leu LeL atctgaaggt ttttcccatt tgaqgaccac tgaggaggaa acaaagataa ggtcaaggaa I ttc ttg i Phe Leu gga ttt aca ctc cta cag tcg tac Gly Phe Thr Leu Leu Gin Ser Tyr caa agg gcc cag aag aaa gga gac Gln Arg Ala Gln Lys Lys Gly Asp 35 a

A

a

I

120 180 240 300 360 420 471 519 567 615 663 ggg tat ggt cca aac Gly Tyr Gly Pro Asn gga ggt ctc ttc cca Gly Gly Leu Phe Pro tta aaa tcg aga ccg gac ata cac ttt gga gta Ile His Phe Gly Val gag gcg aca aaa tgt Glu Ala Thr Lys Cys gcc gcc aag gat cag Ala Ala Lys Asp Gln 50 att cgg tac aat ttt Ile Arg Tyr Asn Phe Asp Leu cga ggc Arg Gly Lys Ser Arg Pro ttc cga tgg ctc Phe Arg Trp Leu cag gcg atg Gin Ala Met ctg ccc aat Leu Pro Asn gtg tcc aag Val Ser Lys 110 ata ttc Ile Phe gca att gaa Ala Ile Glu gag att Glu Ile 85 aac aac agt atg Asn Asn Ser Met act ttc Thr Phe atc Ile acc ctg gga tat Thr Leu Gly Tyr cgc Arg 100 ata ttt gac acg Ile Phe Asp Thr tgt aac acc Cys Asn Thr 105 cag aac aaa Gin Asn Lys 759 807 gcg cta gag gca Ala Leu Giu Ala ctc agc ttt gtg Leu Ser Phe Val atc gac Ile Asp 125 tcg ctg aac tta Ser Leu Asn Leu gag ttc tgt aac Glu Phe Cys Asn tgc Cys 135 tct gac cat atc Ser Asp His Ile 0@ 0 9 0 6 S

S

OS

S

OOSS

S.

@6 0 0

SSS*

6 *506 cca Pro 140 tcc aca ata gca Ser Thr Ile Ala gtg Va1 145 gtc ggg gca acc Vai Giy Ala Thr ggg Gly 150 tca gga atc tcc Ser Giy Ile Ser acg Thr 155 855 903 951 gct gtg gcc aat Ala Val Ala Asn cta Leu 160 ttg gga tta ttt Leu Giy Leu Phe att cca cag gtc Ile Pro Gin Val agc tat Ser Tyr 170 gcc tcc tcg Ala Ser Ser ctg agg acc Leu Arg Thr 190 agc Ser 175 agg ctg ctc agc Arg Leu Leu Ser aac Asn 180 aag aat gag tac Lys Asn Giu Tyr aag gcc ttc Lys Aia Phe 185 atg gcc gag Met Ala Glu 999 1047 atc ccc aat gat Ile Pro Asn Asp gag Glu 195 caa cag gcc acg Gin Gin Ala Thr gcc Ala 200 atc atc Ile Ile 205 gag cac ttc cag Glu His Phe Gin aac tgg gtg gga Asn Trp Vai Gly acc Thr 215 ctg gca gcc gao Leu Ala Ala Asp 0@ OS 0

S

5* 0 C S S 6S gat Asp 220 gac tat ggc cgc Asp Tyr Giy Arg ggc att gac aag Gly Ile Asp Lys ttc Phe 230 cgg gag gag gcc Arg Giu Giu Ala 1095 1143 1191 aag agg gac atc Lys Arg Asp Ile tgt Cys 240 att gac ttc agt Ile Asp Phe Ser atg atc tct cag Met Ile Ser Gin tac tac Tyr Tyr 250 acc cag aag Thr Gin Lys gcc aag gtc Ala Lys Val 270 cag Gin 255 ttg gag ttc atc Leu Giu Phe Ile gac gtc atc cag Asp Val Ile Gin aac tcc tcg Asn Ser Ser 265 gag ccg ctc Glu Pro Leu 1239 1287 atc gtg gtc ttc Ile Val Vai Phe tcc Ser 275 aat ggc ccc gac Asn Gly Pro Asp ctg Leu 280 atc cag Ile Gin 285 gag ata gtt cgg Glu Ile Val Arg aac atc acc gat Asn Ile Thr Asp cgg Arg 295 atc tgg ctg gcc Ile Trp Leu Ala 1335 1383 ago Ser 300 gag got tgg gc Glu Aia Trp Ala ago Ser 305 tot tog otc att Ser Ser Leu Ile gc Ala 310 aag oca gag tao Lys Pro Giu Tyr iii cao gtg gtc ggc ggo aoc ato ggc ttc His Val Val Gly Gly Thr 320 Ile Gly Phe gct Ala 325 ctc agg gog ggg Leu Arg Ala Gly cgt ato Arg Ile 330 1431 cca ggg tto Pro Gly Phe aca atg ggt Thr Met Gly 350 aac Asn 335 aag ttc ctg aag Lys Phe Leu Lys gto cac oca gca Val His Pro Ala ggt cct cgg.

Gly Pro Arg 345 ctg cta ctt Leu Leu Leu 1479 1527 ttg tca agg agt Leu Ser Arg Ser t ct Ser 355 ggg agg aga ctt Gly Arg Arg Leu caa Gin 360 cac oga His Arg 365 gaa gac ctg acg Glu Asp Leu Thr cag Gin 370 otg aag aat tcc Leu Lys Asn Ser aag Lys 375 gtg ccc tog cac Val Pro Ser His gga Gly 380 cog gog gct caa Pro Ala Ala Gin ggg Gly 385 gac ggc tcc aag Asp Giy Ser Lys ggg aac too aga Gly Asn Ser Arg cgg Arg 395 *0 aca goc cta cgc Thr Ala Leu Arg cac His 400 coo tgo act ggg Pro Cys Thr Gly gag Giu 405 gag aac atc aco Giu Asn Ile Thr agc gtg Ser Val 410 1575 1623 1671 1719 1767 gag acc 000 Giu Thr Pro tac gtg gcc Tyr Val Ala 430 ctg gat tat aca Leu Asp Tyr Thr ca o His 420 ctg agg ato tcc Leu Arg Ile Ser tao aat gta Tyr Asn Val 425 atc cac tot Ile His Ser gtc tao too att Val Tyr Ser Ile oao goo otg oaa His Ala Leu Gin tgo aaa Cys Lys 445 000 ggo aog ggo Pro Gly Thr Gly at o Ile 450 ttt goa aao gga Phe Ala Asn Gly tot Ser 455 tgt goa gat att Cys Ala Asp Ile

C

aaa Lys 460 aaa gtt gag gc Lys Val Giu Ala tgg Trp 465 cag gto oto aao Gin Val Leu Asn otg otg oat ctg Leu Leu His Leu 1815 1863 1911 ttt aoo aao ago Phe Thr Asn Ser ggt gag oag gtt Gly Giu Gin Val gao Asp 485 ttt gao gat oaa Phe Asp Asp Gin ggt gao Gly Asp 490 oto aag ggg Leu Lys Gly gaa tog gtg Giu Ser Val 510 aao Asn 495 tao aoo att ato Tyr Thr Ile Ile tgg oag oto too Trp Gin Leu Ser goa gag gat Ala Giu Asp 505 tao got aag Tyr Ala Lys 1959 2007 ttg tto oat gag Leu Phe His Giu gtg Val1 515 ggo aao tao aao Gly Asn Tyr Asn 000 agt Pro Ser 525 gao oga oto aao Asp Arg Leu Asn ato Ile 530 aao gaa aag aaa Asn Giu Lys Lys ato Ile 535 cto tgg agt ggo Leu Trp Ser Gly 2055 2103 too aaa gtg gtt Ser Lys Val Val tto too aao tgo Phe Ser Asn Cys agt Ser 550 oga gao tgt gtg Arg Asp Cys Val oog Pro 555 iv ggc acc agg aag Gly Thr Arg Lys ggg atc Gly Ile 560 atc gag ggg Ile Glu Gly gag ccc acc tgc Glu Pro Thr Cys 565 agt gat gaa aac Ser Asp Glu Asn tgc atg gca Cys Met Ala gcg tgt aca Ala Cys Thr 590 gca gag gga gag Ala Glu Gly Glu tgc ttt gaa Cys Phe Glu 570 gat gca agt Asp Ala Ser 585 aac cac acg Asn His Thr 2151 2199 2247 aag tgc ccg aat Lys Cys Pro Asn ttc tgg tcg aat Phe Trp Ser Asn gag Glu 600 tcg tgc Ser Cys 605 atc gcc aag gag Ile Ala Lys Glu atc Ile 610 gag tac ctg tcg Glu Tyr Leu Ser tgg Trp 615 acg gag ccc ttc Thr Giu Pro Phe ggg Gly 620 atc gct ctg acc Ile Ala Leu Thr atc Ile 625 ttc gcc gta ctg Phe Ala Vai Leu atc ctg atc acc Ile Leu Ile Thr tcc Ser 635 ttc gtg ctg ggg Phe Val Leu Gly gtc Va1 640 ttc atc aag ttc Phe Ile Lys Phe agg Arg 645 aac act ccc atc Asn Thr Pro Ile gtg aag Val Lys 650 gcc acc aac Ala Thr Asn tgc ttc tcc Cys Phe Ser 670 gag ttg tcc tac Glu Leu Ser Tyr ctg Leu 660 ctg ctc ttc tcc Leu Leu Phe Ser ctc atc tgc Leu Ile Cys 665 gac tgg acc Asp Trp Thr 2295 2343 2391 2439 2487 2535 2583 2631 agc tcg ctc atc Ser Ser Leu Ile atc ggc gag ccc Ile Gly Giu Pro tgt cgg Cys Arg 685 ctc cgc caa ccg Leu Arg Gin Pro gcc Ala 690 ttt ggc atc agc Phe Gly Ile Ser ttc Phe 695 gtc ctg tgc atc Vai Leu Cys Ile tcc Ser 700 tgc atc ctg gtg Cys Ile Leu Vai aag Lys 705 acc aac cgg gtg Thr Asn Arg Vai ctg gtc ttc gag Leu Val Phe Glu gcc Ala 715 aag atc ccc acc Lys Ile Pro Thr agc Ser 720 ctc cac cgc aag Leu His Arg Lys tgg Trp 725 gtg ggc ctc aac Val Giy Leu Asn ctg cag Leu Gin 730 ttc ctc ctg Phe Leu Leu atc tgg ctc Ile Trp Leu 750 ttc ctc tgc atc Phe Leu Cys Ile ctg Leu 740 gtg caa atc gtc Vai Gin Ile Val acc tgc atc Thr Cys Ile 745 cat gag ctg His Giu Leu 2679 2727 tac acc gcg cct Tyr Thr Aia Pro tcc agc tac agg Ser Ser Tyr Arg gag gac Glu Asp 765 gag gtc atc ttc Glu Val Ile Phe atc Ile 770 acc tgc gac gag Thr Cys Asp Glu ggc tcg ctc atg Gly Ser Leu Met 775 gcc gcc atc tgc Ala Aia Ile Cys gcg Ala ttc Phe 795 2775 2823 ctg Leu 780 ggc ttc ctc atc Gly Phe Leu Ile tac acc tgc ctc Tyr Thr Cys Leu ctc Leu 790 ttc ttc gcc ttc aag tcc cgt aag ctg ccg gag aac ttc aac gag gct 2871 Phe Phe Ala Phe Lys 800 Ser Arg Lys Leu Pro Glu 805 Asn Phe Asn Glu Ala 810 aag ttc atc Lys Phe Ile ttc atc coo Phe Ile Pro 830 ac Thr 815 ttc agc atg ttg Phe Ser Met Leu atc Ile 820 ttc ttc ato gtc Phe Phe Ile Val tgg ato too Trp Ile Ser 825 tcg gcc gtg Ser Ala Val 2919 2967 gcc tat gtc agc Ala Tyr Val Ser acc Thr 835 tac ggo aag ttt Tyr Gly Lys Phe gtg Va1 840 gag gtg Glu Val 845 att gcc atc ctg Ile Ala Ile Leu tco ago tto ggg Ser Ser Phe Gly ctg Leu 855 ctg ggc tgc att Leu Gly Cys Ile ttc aac aag tgt Phe Asn Lys Cys atc atc ctg ttc Ile Ile Leu Phe aag Lys 870 cog tgo cgt aao Pro Cys Arg Asn ato gag gag gtg Ile Giu Giu Val cgo Arg 880 tgo ago acg gcg Cys Ser Thr Ala goo Ala 885 oac goo tto aag His Ala Phe Lys gtg gcg Val Ala 890 goc ogg goo Ala Arg Ala ago otg tgc Ser Leu Cys 910 ac Thr 895 otc ogg cgo ago Leu Arg Arg Ser gcg tot cgo aag Ala Ser Arg Lys cgo too ago Arg Ser Ser 905 aco tgc ggg Thr Cys Gly 3015 3063 3111 3159 3207 3255 3303 3351 ggo too aoo ato Gly Ser Thr Ile too Ser 915 tcg coo goo tcg Ser Pro Ala Ser too Ser 920 ccg ggo Pro Gly 925 otc aoo atg gag Leu Thr Met Glu atg Met 930 oag cgo tgc ago Gin Arg Cys Ser acg Thr 935 cag aag gto ago Gin Lys Val Ser ggo ago ggo aoo Gly Ser Gly Thr aco ctg tog otc Thr Leu Ser Leu ago Ser 950 tto gag gag aca Phe Glu Giu Thr ggo Gly 955 oga tao gc aco Arg Tyr Ala Thr otc Leu 960 ago cgo aog gc Ser Arg Thr Ala cgo Arg 965 ago agg aao tcg Ser Arg Asn Ser gcg gat Ala Asp 970 ggo cgc ago Gly Arg Ser cct cag aaa Pro Gin Lys 990 ggo Gly 975 gao gao ctg oca Asp Asp Leu Pro aga cac cac gac Arg His His Asp tgo gag cco cag Cys Giu Pro Gin CcC Pro 995 gc aac gat Ala Asn Asp goc oga Ala Arg 1000 oag ggo cog Gin Gly Pro 985 tao aag gcg Tyr Lys Ala aag gag cgo Lys Giu Arg 3399 3447 gcg ccg Ala Pro 1005 coo aca Pro Thr 1020 aco aag ggc Thr Lys Gly aco cta Thr Leu 1010 gag tog oog Glu Ser Pro ggc ggo ago Gly Gly Ser 1015 3495 act atg gag gaa acc Thr Met Giu Giu Thr 1025 taa tcoaaotcot coatoaacco caagaacato 3549 otccacggca gcaccgtoga oaactgacat oaactcctaa ocggtggotg cccaacctct 3609 vi cccctctccg tgattttctg acaattaggt t tatt ct ct c attgtcaaga cactgtgatg tataatgact tqtaaaattg aaaaaaaaaa qcactttqcg acttggatat gagcagagtt gaattgtatt taatttgtta acagaactgt qtaacaaaaa gtaattactt aaaaaaaaaa ttttgctgaa ttactagtgt gtgtcaaaqt acaaacattt caacatataa tttataacat aattqttgat ctgtacatta aaaagcggcc gattqcagca gcgatggaat atctgaacta qaagtatttt gqtaccacct ttatcattga tatcttaaaa aatgcatatt cgacagcaac tctgcagttc atcacaacat tctgaagtat tagtgacatt gaagcagtqa aacctggatt atgcaaattg tcttgataaa gq cttttatccc aatgagttgc ctgaactact atgttctaac ctgagattgc gcaacaggaa taatcagatg aaaaaaaaaa 3669 3729 3789 3849 3909 3969 4029 4089 4131 <210> 2 <211> 1026 <212> PRT <213> Squalus acanthias <400> 2 Met Ala Gin Leu His Cys 1 Gin Ser Tyr Asn Val Ser Gin Leu Leu Phe Leu Gly Phe Thr Leu Leu Gly Tyr Gly Pro Leu Phe Asn Gin Arg Lys Gly Asp Ala Ala Lys Ile Ile Leu Gly Gly Lys Pro Ile Ala Gin Lys Phe Gly Vai Thr Lys Cys Asp Gin Asp Ile Arg Leu 55 Gly Ser Arg Pro Giu Gin Tyr Asn Phe Arg 70 As n Phe Arg Trp Leu 75 Phe Ala Met Ile Phe Ile Giu Glu Asn Ser Met Thr Asn Leu Pro Asn Ile Thr Leu Gly Tyr Giu Ala Thr 115 Leu Asp Giu Arg 100 Leu Phe Asp Thr Thr Val Ser Ser Phe Val Ala 120 Ser Asn Lys Ile Asp 125 Ser Lys Ala Leu 110 Ser Leu Asn Thr Ile Ala Phe Cys Asn Asp His Ile 130 Val Val Pro 140 Al a Gly Ala Thr 145 Leu Gly 150 Ile Gly Ile Ser Val Ala Asn Leu 160 Arg Gly Leu Phe Tyr 165 Pro Gin Val Ser 170 Ala Ser Ser Leu Leu Ser Asn Lys Asn Giu Tyr Lys Ala Phe Leu Arq Thr Ile Pro 180 185 190 vii Asn Asp Giu Gin Gin Ala Thr 195

M

0* Gin Pro 225 Ile Glu Vai Arg Ser 305 Thr Phe Arg Thr Giy 385 Pro Asp Ser Giy Trp 465 Gly Thr Trp 210 Giy Asp Phe Phe Arg 290 Ser Ile Leu Ser Gin 370 Asp Cys T yr Ile Ile 450 Gin Giu Ile As r Ile Phe Ile Ser 275 Asn Ser Gi y Lys Ser 355 Leu Giy Thr Thr Ala 435 Phe Vali Gin Ile Trp Asp Ser Al a 260 Asn Ile Leu Phe Giu 340 Gly Lys Ser Giy His 420 His Ala Leu Vai Asn 500 Val Lys Giu 245 Asp Gly Thr Ile Al a 325 Val1 Arg Asn Lys Glu 405 Leu Al a As n Asn Asp 4185 rrp Gly Phe 230 Met Val1 Pro Asp Al a 310 Leu His Arg Ser Ala 390 Glu Arg Leu Gly His 470 Phe Gin Thr 215 Arg Ile Ile Asp Arg 295 Lys Arg Pro Leu Lys 375 Gly Asn Ile Gin Ser 455 Leu Asp Leu Ala Met Ala 200 Leu Ala Ala Giu Giu Ala Ser Gin Tyr 250 Gin Asn Ser 265 Leu Giu Pro 280 Ile Trp Leu Pro Giu Tyr Ala Gly Arq 330 Ala Gly Pro 345 Gin Leu Leu 360 Val Pro Ser Asn Ser Arg Ile Thr Ser 410 Ser Tyr Asn 425 Asp Ile His 440 Cys Ala Asp Leu His Leu Asp Gin Gly 490 Ser Ala Giu 505 Glu Asp Val 235 Tyr Ser Leu Ala Phe 315 Ile Arg Leu His Arg 395 Val Val Ser Ile Lys 475 Asp A\sp Sle Asp 220 Lys Thr Al a Ile Ser 300 His Pro Thr His Gly 380 Thr Giu Tyr Cys Lys 460 Phe Leu Glu Ile 205 Asp Arg Gin Lys Gin 285 Giu Val1 Gly Met Arg 365 Pro Al a Thr Val1 Lys 445 Lys Thr Lys Ser Glu Tyr Asp Lys Val1 270 Giu Al a Val1 Phe Gly 350 Giu Ala Leu Pro Ala 430 Pro Val Asn Gly Val 510 His Gly Ile Gin 255 Ile Ile Trp Gi y As n 335 Leu Asp Al a Arg Tyr 415 Val1 Gly Glu Ser Isn 4 Leu Phe Arq Cys 240 Leu Val1 Val1 Al a Gly 320 Lys Ser Leu Gin His 400 Leu Tyr Thr Al a Met 480 Tyr Phe His Giu Val Gly Asn Tyr Asn Ala Tyr Ala Lys Pro Ser Asp Arg Leu viii Asn Ile Asn Giu Lys Lys Ile Leu Trp Ser S. S Pro 545 Ile Giu Pro Giu Ile 625 Phe Leu Leu Pro Lys 705 Leu Leu Ala Phe Giy 785 Ser Ser 530 Phe Ile Giy Asn Ile 610 Phe Ile Ser Ile Ala 690 Thr His Cys Pro Ile 770 Tyr Arg Met Ser Giu Giu Asp 595 Giu Aila Lys Tyr Phe 675 Phe Asn Arq Ile Pro 755 I'hr Thr Lys Leu Asn Giy Phe 580 Phe Tyr Val1 Phe Leu 660 Ile Giy Arg Lys Leu 740 Ser Cys Cys Leu Ile 820 Cys Giu 565 Ser Trp Leu Leu Arg 645 Leu Giy Ile Vali Trp 725 Vali Ser Asp Leu Pro 805 Phe Ser 550 Pro Asp Ser Ser Giy 630 Asn Leu Glu Ser Leu 710 Vali Gin Tyr Giu Leu 790 Giu Phe 535 Arq Thr Giu As n Trp 615 Ile Thr Phe Pro Phe 695 Leu Giy Ile Arg Gly 775 Al a As n Ile Asp Cys Asn Giu 600 Thr Leu Pro Ser Arg 680 Vali Vai Leu Vai Asn 760 Ser Aia Phe Val Cys Cys Asp 585 Asn Giu Ile Ile Leu 665 Asp Leu Phe Asn Thr 745 His Leu Ile As n Trp 825 Val1 Phe 570 Aila His Pro Thr Vali 650 Ile Trp Cys Giu Leu 730 Cys Giu Met Cys Giu 810 Ile Giy Pro 555 Giu Ser Thr Phe Ser 635 Lys Cys Thr Ile Aia 715 Gin Ile Leu Aia Phe 795 Aia Ser Phe 540 Gly Cys Aila Ser Giy 620 Ph e Aila Cys Cys Ser 700 Lys Phe Ile G1u Leu 780 Phe Lys Phe Ser Thr Met Cys Cys 605 Ile Vali Thr Phe Arg 685 Cys Ile Leu Trp Asp 765 Giy Phe Phe Ile Lys Arg Aia Thr 590 Ile Aia Leu As n Ser 670 Leu Ile Pro Leu Leu 750 Giu Phe Ala Ile Pro 830 Val Lys Cys 575 Lys Ala Leu Gly Arg 655 Ser Arg Leu Thr Vai 735 Tyr Vali Leu Phe Thr 815 5i a -Vai Gly 560 Aia Cys Lys Thr Vali 640 Giu Ser Gin Val Ser 720 Phe Thr Ile Ile Lys 800 Phe T yr Vai Ser Thr Tyr Gly Lys Phe Val Ser Ala Vai Giu Vai Ile Ala Ile 835 840 845 ix Leu Ala Ser Ser Phe Gly Leu Leu Gly Cys Ile 850 855 Tyr 860 Ile Phe Asn Lys Cys Tyr 865 Cys Ile Ile Leu Phe Lys 870 His Pro Cys Arg Asn Thr 875 Ala Giu Giu Val Arg 880 Ser Thr Ala Al a 885 Ala Ala Phe Lys Val1 890 Ser Ala Arg Ala Thr Leu 895 Arq Arq Ser Thr Ile Ser 915 Giu Met Gin Ser Arg Lys Arg 905 Thr Ser Ser Leu Pro Ala Ser Ser 920 Gin Cys Gly Pro Gly 925 Gly Cys Gly Ser 910 Leu Thr Met Ser Gly Thr Arq Cys Ser 930 Val Thr Thr 935 Phe Lys Val Ser Phe 940 Arg Leu Ser Leu 945 Ser Ser 950 Ser Giu Giu Thr Gly 955 Asp Tyr Ala Thr Leu 960 Asp Arg Thr Ala Arg 965 Arg Arg Asn Ser Ala 970 Gly Giy Arg Ser Gly 975 Asp Leu Pro Pro Gin Pro 995 His His Asp Gin 985 Tyr Pro Pro Gin Lys Cys Giu 990 Thr Lys Gly Asn Asp Ala Arg 1000 Lys Ala Ala Pro 1005 Thr Leu Giu Ser Pro Gly Gly Ser Lys Giu Arg Pro Thr Thr Met Giu

S

1010 Giu Thr 025 1015 1020 <210> 3 <211> 4134 <212> DNA <213> Squalus acanthias <220> <221> CDS <222> (439)..(3522) <400> 3 aattccqttg ctgtcggttc agtccaagtc gccattacaq gaacatgcac tacatctgtg tattaaaatg tttctgcaag gatggcttca gtcattgtat gaataactga ccaaagggat gttcaccctt tcttgqagca tacgatcaac atggqqattg atcttccagg aqttctgctq gcagaaatcc tccaggcatc ctctgtaaac tcctccagtg ttaatgaaat cqagaaatca qtaacaaaat cctgaaggaq taaagcgatc gggctggcgt caaaatgaga attgtcagtt attctqcacg ggaacaaagc atggaaqact cctcaccatt agtgtgqctt aatggtqgtc atctgaaggt ttttcccatt tgaggaccac tgaggaggaa acaaagataa ggtcaaggaa 120 180 240 300 360 420 cagagacagg gctgcaca atg gct cag ctt Met Ala Gin Leu 1 cac tgc caa ctc tta ttc ttg His Gys Gin Leu Leu Phe Leu gga ttt aca ctc cta cag tcg tac Gly Phe Thr Leu Leu Gin Ser Tyr aat Asn 20 gtc tca ggg tat Val Ser Gly Tyr ggt cca aac Gly Pro Asn ctc ttc cca Leu Phe Pro 519 caa agg gcc Gin Arg Ala cag aaq aaa gga Gin Lys Lys Gly gac Asp 35 atc ata ctg gga Ile Ile Leu Gly ggt Gly ata cac Ile His ttt gga gta gec Phe Gly Val Ala gcc Ala 50 aag gat cag gac Lys Asp Gin Asp tta Leu aaa tcg aga ccg Lys Ser Arg Pro gag Glu 60 gcg aca aaa tgt Ala Thr Lys Cys att Ile 65 cgg tac aat ttt Arg Tyr Asn Phe oga Arg 70 ggc ttc cga tgg Gly Phe Arg Trp ctc Leu r cag gcg atg ata Gin Ala Met Ile gca att gaa gag Ala Ile Giu Glu att Ile aac aac agt atg Asn Asn Ser Met act ttc Thr Phe 615 663 711 759 807 ctg ccc aat Leu Pro Asn gtg tcc aag Val Ser Lys 110 atc Ile acc ctg gga tat Thr Leu Gly Tyr cgc Arg 100 ata ttt gac acg Ile Phe Asp Thr tgt aac acc Cys Asn Thr 105 cag aac aaa Gin Asn Lys gcg cta gag gca Ala Leu Giu Ala ctc agc ttt gtg Leu Ser Phe Val

S

S.

S.

atc gac Ile Asp 125 tcg ctg aac tta Ser Leu Asn Leu gat Asp 130 gag ttc tgt aac Glu Phe Cys Asn tgc Cys 135 tct gac cat atc Ser Asp His Ile cca Pro 140 tcc aca ata gca Ser Thr Ile Ala gtg Val 145 gtc ggg gca acc Val Gly Ala Thr tca gga atc tcc Ser Gly Ile Ser acg Thr 155 855 903 951 gct gtg gcc aat Ala Val Ala Asn cta Leu 160 ttg gga tta ttt Leu Giy Leu Phe tac Tyr 165 att cca cag gtc Ile Pro Gin Val agc tat Ser Tyr .170 gcc tcc tcg Ala Ser Ser ctg agg acc Leu Arg Thr 190 agg ctg ctc agc Arg Leu Leu Ser aac Asn 180 aag aat gag tac Lys Asn Giu Tyr aag gcc ttc Lys Ala Phe 185 atg gcc gag Met Ala Glu 999 1047 atc ccc aat gat Ile Pro Asn Asp caa cag gcc acg Gin Gin Ala Thr atc atc Ile Ile 205 gag cac ttc cag Glu His Phe Gin tgg Trp 210 aac tgg gtg gga Asn Trp Val Gly acc Thr 215 ctg gca gcc gac Leu Ala Ala Asp 1095 1143 gat Asp 220 gac tat ggc cgc Asp Tyr Giy Arg cca Pro 225 ggc att gac aag Gly Ile Asp Lys cgg gag gag qcc Arg Giu Glu Ala gtt Va1 235 -xi aag agg gac atc Lys Arg Asp Ile tgt Cys 240 att gac ttc agt Ile Asp Phe Ser gag Glu 245 atg atc tct cag Met Ile Ser Gin tac tac Tyr Tyr 250 1191 acc cag aag Thr Gin Lys gcc aag gtc Ala Lys Val 270 cag Gin 255 ttg gag ttc ate Leu Glu Phe Ile gcc Ala 260 gac gtc ate Asp Val Ile cag aac tcc tcg Gin Asn Ser Ser 265 1239 1287 atc gtg gtc ttc Ile Val Val Phe tecc Ser 275 aat ggc ccc gac Asn Gly Pro Asp ctg Leu 280 gag ccg ctc Glu Pro Leu atc cag Ile Gin 285 gag ata gtt cgg Glu Ile Val Arg aga Arg 290 aac atc acc gat Asn Ile Thr Asp cgg Arg 295 ate tgg ctg gcc Ile Trp Leu Ala agc Ser 300 gag gct tgg gcc Glu Ala Trp Ala tct tcg ctc att Ser Ser Leu Ile gcc Ala 310 aag cca gag tac Lys Pro Glu Tyr ttc Phe 315

S

S

S.

cac gtg gtc ggc His Val Val Gly ggc Gly 320 acc ate ggc ttc Thr Ile Gly Phe get Ala 325 ctc agg gcg ggg Leu Arg Ala Gly cgt ate Arg Ile 330 1335 1383 1431 1479 1527 cca ggg ttc Pro Gly Phe gac aat ggg Asp Asn Gly 350 aac Asn 335 aag ttc ctg aag Lys Phe Leu Lys gag Glu 340 gtc cac ccc ago Val His Pro Ser agg tec tcg Arg Ser Ser 345 aac tgc tac Asn Cys Tyr ttt gtc aag gag Phe Val Lys Glu tgg gag gag acc Trp Glu Glu Thr ttc acc Phe Thr 365 gag aag acc ctg Glu Lys Thr Leu cag ctg aag aat Gin Leu Lys Asn tecc Ser 375 aag gtg ccc tcg Lys Val Pro Ser cac His 380 gga ccg gcg gct Gly Pro Ala Ala caa Gin 385 ggg gac ggc tec Gly Asp Gly Ser gcg ggg aac tec Ala Gly Asn Ser aga Arg 395 1575 1623 1671 cgg aca gcc cta Arg Thr Ala Leu cgc Arg 400 cac ccc tgc act His Pro Cys Thr ggg Gly 405 gag gag aac atc Glu Glu Asn Ile acc age Thr Ser 410 gtg gag acc Val Glu Thr gta tac gtg Val Tyr Val 430 tac ctg gat tat Tyr Leu Asp Tyr.

aca Thr 420 cac ctg agg atc His Leu Arg Ile tcc tac aat Ser Tyr Asn 425 gac ate cac Asp Ile His 1719 1767 gcc gtc tac tec Ala Val Tyr Ser gct cac gcc ctg Ala His Ala Leu caa Gin 440 tct tgc Ser Cys 445 aaa ccc ggc acg Lys Pro Gly Thr ggc Gly 450 atc ttt gca aac Ile Phe Ala Asn gga Gly 455 tct tgt gca gat Ser Cys Ala Asp 1815 1863 att Ile 460 aaa aaa gtt gag Lys Lys Val Glu gcc Ala 465 tgg cag gtc ctc Trp Gin Val Leu aac Asn 470 cat ctg ctg cat His Leu Leu His ctg Leu 475 aag ttt acc aac agc atg ggt gag cag gtt gac ttt gac gat caa ggt 1911 -xii- Lys Phe Thr gac otc aag Asp Leu Lys gat gaa tcg Asp Giu Ser 510 Asn Ser 480 Met Gly Giu Gin Val Asp Phe Asp 485 aao tgg oag ctc Asn Trp Gin Leu aac tac acc att Asn Tyr Thr Ile atc Ile 500 Asp Gin Gly 490 tco gca gag Ser Ala Glu 505 goc tac gct Ala Tyr Ala 1959 2007 gtg ttg ttc cat Val Leu Phe His gag Glu 515 gtg ggc aac tao Val Gly Asn Tyr aac Asn 520 aag coo Lys Pro 525 agt gac cga ctc Ser Asp Arg Leu atc aac gaa aag Ile Asn Giu Lys aaa Lys 535 atc otc tgg agt Ile Leu Trp Ser ggc Gly 540 tto too aaa gtg Phe Ser Lys Val cct tto too aao Pro Phe Ser Asn tgc Cys 550 agt oga gao tgt Ser Arg Asp Cys gtg Val 555 ocg ggo aoo agg Pro Gly Thr Arg aag Lys 560 ggg ato ato gag Gly Ile Ile Glu ggg Gly 565 gag ccc aoo tgo Glu Pro Thr Cys tgo ttt Cys Phe 570 gaa tgo atg Glu Cys Met agt gog tgt Ser Ala Cys 590 gca Ala 575 tgt goa gag gga Cys Ala Glu Gly gag Glu 580 tto agt gat gaa Phe Ser Asp Glu aao gat gca Asn Asp Ala 585 gag aac cac Glu Asn His 2055 2103 2151 2199 2247 2295 2343 2391 aca aag tgo ccg Thr Lys Cys Pro aat Asn 595 gat tto tgg tcg Asp Phe Trp Ser acg tcg Thr Ser 605 tgo ato goo aag Cys Ile Ala Lys gag Glu 610 ato gag tac ctg Ile Giu Tyr Leu tcg Ser 615 tgg aog gag cc Trp Thr Giu Pro tto Phe 620 ggg ato got otg Gly Ile Ala Leu ato tto goo gta Ile Phe Ala Val ggo ato ctg ato Gly Ile Leu Ile ac Thr 635 too tto gtg otg Ser Phe Val Leu ggg Gly 640 gto tto ato aag Val Phe Ile Lys agg aao act 000 Arg Asn Thr Pro ato gtg Ile Val 650 aag goc aoo Lys Ala Thr tgo tgo tto Cys Cys Phe 670 aao Asn 655 cgg gag ttg too Arg Giu Leu Ser tao Tyr 660 otg otg otc tto Leu Leu Leu Phe too otc ato Ser Leu Ile 665 agg gao tgg Arg Asp Trp 2439 2487 too ago tog otc Ser Ser Ser Leu ato Ile 675 tto ato ggo gag Phe Ile Gly Glu ac tgt Thr Cys 685 ogg otc cgo caa Arg Leu Arg Gin gc ttt ggo ato Ala Phe Giy Ile ago Ser 695 tto gto ctg tgo Phe Val Leu Cys 2535 2583 ato Ile 700 too tgo ato ctg Ser Cys Ile Leu gtg Va1 705 aag aco aac ogg Lys Thr Asn Arg otg otg gtc tto Leu Leu Val Phe goo aag ato cc aco ago otc cac cgo aag tgg gtg ggo otc aac ctg Ala Lys Ile Pro Thr Ser Leu His Arg Lys Trp Val Gly Leu Asn Leu 2631 -xiii- 730 cag tto ctc Gin Phe Leu ato atc tgg Ile Ile Trp 750 gtc ttc otc tgc Vai Phe Leu Cys atc Ile 740 otg qtg oaa atc Leu Vai Gin Ile gtc acc tgc Vai Thr Cys 745 aac cat gag Asn His Glu 2679 2727 ctc tac acc gcg Leu Tyr Thr Ala cc too agc tao Pro Ser Ser Tyr ctg gag Leu Glu 765 gao gag gtc atc Asp Giu Val Ile ato acc tgc gac Ile Thr Cys Asp gag Glu 775 ggc tog otc atg Gly Ser Leu Met gcg Ala 780 otg ggc ttc otc Leu Giy Phe Leu ato Ile 785 ggo tao aco tgo Gly Tyr Thr Cys otc gcc gc ato Leu Ala Aia Ile tgo Cys 795 tto tto tto goo Phe Phe Phe Ala tto Phe 800 aag too cgt aag Lys Ser Arg Lys otg Leu 805 ccg gag aao tto Pro Giu Asn Phe r r r aao gag Asn Glu 810 got aag tto Ala Lys Phe too tto ato Ser Phe Ile 830 ato Ile 815 ac tto ago atg Thr Phe Ser Met ttg Leu 820 ato tto tto ato Ile Phe Phe Ile gto tgg ato Vai Trp Ile 825 gtg tog goo Vai Ser Ala 2775 2823 2871 2919 2967 3015 3063 3111 ccc goo tat gto Pro Ala Tyr Vai ago Ser 835 ac tao ggo aag Thr Tyr Giy Lys gtg gag Vai Glu 845 gtg att goo ato Val Ile Ala Ile otg Leu 850 goo too ago tto Ala Ser Ser Phe ggg Gly 855 ctg otg ggo tgo Leu Leu Giy Cys tao tto aao aag Tyr Phe Asn Lys tao ato ato ctg Tyr Ile Ile Leu aag ccg tgc cgt Lys Pro Cys Arg ac ato gag gag Thr Ile Giu Glu gtg Vai 880 cgc tgo ago acg Arg Cys Ser Thr gc cac goo tto Ala His Ala Phe aag gtg Lys Val 890 gcg goo ogg Ala Ala Arg ago ago ctg Ser Ser Leu 910 goo Ala 895 ac otc ogg cgo Thr Leu Arg Arg ago Ser 900 goo gog tot cgo Ala Ala Ser Arg aag cgo too Lys Arg Ser 905 too aoo tgo Ser Thr Cys 3159 3207 tgo ggo too aoo Cys Gly Ser Thr ato Ile 915 too tog cc goo Ser Ser Pro Ala ggg oog Gly Pro 925 ggo otc aoo atg Gly Leu Thr Met atg oag cgo tgo Met Gin Arg Cys ago Ser 935 acg oag aag gto Thr Gin Lys Val ago Ser 940 tto ggo ago ggo Phe Gly Ser Gly ac Thr 945 gto aoo ctg tcg Val Thr Leu Ser ago tto gag gag Ser Phe Giu Glu 3255 3303 3351 ggo oga tao goo Gly Arg Tyr Ala ac Thr 960 otc ago cgo acg Leu Ser Arg Thr goo Ala 965 cgo ago agg aao Arg Ser Arg Asn tcg gcg Ser Ala 970 xiv gat ggc cgc Asp Gly Arg cog cot cag Pro Pro Gin 990 gog gog cog Ala Ala Pro ago Se r 975 aaa Lys ggo gao gao Gly Asp Asp tgo gag ooo Cys Glu Pro ot g Leu tot aga cao oao Ser Arg His His gao oag ggo Asp Gin Gly 985 1005 ogo coo aco aag ggo aoo Thr Lys Gly Thr 1010 act atg gag gaa Thr Met Giu Giu cag coo goc aac gat goc oga t Gin Pro Ala Asn Asp Ala Arg T 995 1000 ota gag tog cog gqc ggo ago a Leu Giu Ser Pro Gly Gly Ser L 1015 aco taa tcoaaotcot ccatoaaooo Thr ac aag yr Lys ag gag ys Giu 3399 3447 3495 aca 3542 Arg Pro Thr 1020 1 L025 caagaacatc cccaaootot ottttatoco aatgagttgc ot ga act act atgttotaac otgagattgc gcaacaggaa taatcagatg aaaaaaaaaa otocacggoa ccctotoog tgattttctg acaattaggt ttattctctc attgtcaaga oaotgtgatg tataatgaot tgtaaaattg aaaaaaaaaa goaoogt oga gcaotttgcg acttggatat gagoagagtt gaattgtatt taatttgtta aoagaaotgt gtaacaaaaa gtaattactt aaaaaaaaaa oaaotgaoat ttttgctgaa ttaotagtgt gtgtoaaagt aoaaacattt caacatataa tttataaoat aattgttgat otgtaoatta aaaagcggoc oaactcotaa gattgcagca gcgatggaat atotgaacta gaagtatttt gqtaccacct ttatoattga tatottaaaa aatgcatatt cgacagcaac ccggtggctg tot gcagt to atoaoaaoat tctgaagtat tagtgacatt gaagoagtga aacctggatt atgoaaattg tottgataaa gg 3602 3662 3722 3782 3842 3902 3962 4022 4082 4134 <210> 4 <211> 1027 <212> PRT <213> Squalus acanthias <400> 4 Met Ala Gin Leu His Cys 1 5 Gin Ser Tyr Asn Val Ser Lys Gly Asp Ile Ile Leu Ala Ala Lys Asp Gin Asp Ile Arg Tyr Asn Phe Arg 70 Ala Ile Glu Glu Ile Asn Gin Leu Leu Giy Tyr Giy 25 Giv Gly Leu Leu Gly Phe Thr Leu Leu Asn Gin Arg Phe Pro Ile Aia Gin Lys Phe Giy Val Thr Lys Cys Leu Lys Ser Arg Pro 55 Gly Giu Gin Phe Arg Trp Leu Ala Met Ile Asn Ser Met Phe Leu Pro Asn xv Leu Giy Tyr Arg Ile Phe Asp Thi 100 *r 0 .0 0 0 0* 0* Glu Leu Va1 145 Leu Leu Asn Gin Pro 225 Ile Glu Vai Arg Ser 305 Thr Phe Lys Leu Gin 385 Alz Asp 130 Va1 Gly Leu Asp Trp 210 Gly Asp Phe Phe Arg 290 Ser Ile Leu lu rhr 370 ,ly Thr 115 Glu Gly Leu Ser Glu 195 Asn Ile Phe Ile Ser 275 Asn Ser Gly Lys Phe 355 Gin Asp Let Phe Ala Phe Asn 180 Gin Trp Asp Ser Ala 260 Asn Ile Leu Phe lu 340 Frp Leu 31Y i Ser Cys Thr Tyr 165 Lys Gin Va1 Lys Glu 245 Asp Gly Thr Ile Ala 325 Val Glu Lys Ser PhE Asr Gly 150 Ile Asn Ala Gly Phe 230 Met Va1 Pro Asp Ala 310 Leu His lu ,sn Lys 390 Vai Cys 135 Ser Pro Glu Thr Thr 215 Arg Ile Ile Asp Arg 295 Lys Arg Pro Thr Ser I 375 Ala Alz 120 Ser Gli Gin Tyr Ala 200 Leu Glu Ser Gin Leu 280 Ile Pro Ala Ser Phe 360 Lys ;ly Cys Asn 105 Gin Asn Asp His Ile Ser Vai Ser 170 Lys Ala 185 Met Ala Ala Ala Glu Ala Gin Tyr 250 Asn Ser 265 Glu Pro Trp Leu Glu Tyr Gly Arg 330 Arg Ser 345 Asn Cys Val Pro Asn Ser I Thi Lys Ile Thr 155 Tyr Phe Glu Asp Vai 235 Tyr Ser Leu Ala Phe 315 Ile Ser Cyr 3er ~rg 395 Val Ile Pro 140 Ala Ala Leu Ile Asp 220 Lys Thr Ala Ile Ser 300 His Pro Asp I Phe 'I His C 380 Arg T Se As 12E Sel Val Sex Arg Ile 205 Asp Arg Gin Lys ln 285 lu lal ~sn 'hr 365 ily hr r Lys 110 3 Ser Thr Ala Ser Thr 190 Glu Tyr Asp Lys Va1 270 Glu Ala Val Phe I Gly 1 350 Glu I Pro P Ala L Al Lei IlE Asr Sex 175 Ile His Gly Ile Gin 255 Ile Ile Trp Isn 335 'he lys la leu a Leu a Asn I Ala 1 Leu 160 Arg Pro Phe Arg Cys 240 Leu Va1 Va1 Ala Gly 320 Lys Va1 Thr Ala Arg His Pro Cys Thr Gly Giu Giu Asn le Thr Ser Val Giu Thr Pro Tyr 405 410 415 xvi Leu Asp Tyr Thr His Leu Arg Ile Ser Tyr Asn Val Tyr Val Ala Val 420 425 430 Tyr Ser Ile Ala His Ala Leu Gin Asp Ile His Ser Cys Lys Pro Gly 435 440 445 Thr Gly Ile The Ala Asn Gly Ser Cys Ala Asp Ile Lys Lys Val Glu 450 455 460 Ala Trp Gin Val Leu Asn His Leu Leu His Leu Lys Phe Thr Asn Ser 465 470 475 480 Met Gly Giu Gin Val Asp Phe Asp Asp Gin Gly Asp Leu Lys Gly Asn 485 490 495 Tyr Thr Ile Ile Asn Trp Gin Leu Ser Ala Giu Asp Glu Ser Val Leu 500 505 510 Phe His Giu Val Gly Asn Tyr Asn Ala Tyr Ala Lys Pro Ser Asp Arq *515 520 525 *Leu Asn Ile Asn Giu Lys Lys Ile Leu Trp Ser Gly Phe Ser Lys Val 530 535 540 *9Vai Pro Phe Ser Asn Cys Ser Arq Asp Cys Val Pro Gly Thr Arg Lys *545 550 555 560 Gly Ile Ile Giu Gly Glu Pro Thr Cys Cys Phe Giu Cys Met Ala Cys 565 570 575 *Ala Giu Gly Giu Phe Ser Asp Giu Asn Asp Ala Ser Ala Cys Thr Lys 580 585 590 Cys Pro Asn Asp Phe Trp Ser Asn Glu Asn His Thr Ser Cys Ile Ala 595 600 605 Lys Giu Ile Giu Tyr Leu Ser Trp Thr Glu Pro Phe Gly Ile Ala Leu 610 615 620 :Thr Ile Phe Ala Val Leu Gly Ile Leu Ile Thr Ser Phe Val Leu Gly .625 630 635 640 Val Phe Ile Lys Phe Arq Asn Thr Pro Ile Val Lys Ala Thr Asn Arg 645 650 655 Giu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Ile Cys Cys Phe Ser Ser 660 665 670 Ser Leu Ile Phe Ile Giy Glu Pro Arq Asp Trp Thr Cys Arg Leu Arg 675 680 685 Gin Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys Ile Leu 690 695 700 Val Lys Thr Asn Arg Val Leu Leu Val Phe Giu Ala Lys Ile Pro Thr 705 710 715 720 Ser Leu His Arg Lys Trp Val Gly Leu Asn Leu Gin Phe Leu Leu Val 725 730 735 Phe Leu Cys Ile Leu Val Gin Ile Val Thr Cys Ile Ile Trp Leu Tyr xvii Thr Ala Pro r e c Ile Ile 785 Lys Phe Tyr Ile Cys 865 Arg Leu Ser Met Thr 945 Leu Asp Glu Phe 770 Gly Ser Ser Val Leu 850 Tyr Cys Arg Thr Glu 930 Val Ser Asp Pro 755 Ile Tyr Arg Met Ser 835 Ala Ile Ser Arg Ile 915 Met Thr Arg Leu Gln 995 740 SPro SThr Thr Lys Leu 820 Thr Ser Ile Thr Ser 900 Ser Gln Leu Thr Pro 980 Pro 745 Asn Ser Ser Tyr Arg His Glu Leu Glu Cys Cys Leu 805 Ile Tyr Ser Leu Ala 885 Ala Ser Arg Ser Oda 965 Ser Ila Asp Leu 790 Pro Phe Gly Phe Phe 870 Ala Ala Pro Cys Leu 950 Arg Arg Asn Gl 77 Le G1 Ph Ly G1 85 Ly Hi: Sei Ali Se~ 93 Sei Sei His

ISE

.u Gly 5 *u Ala Asn Ile Phe 840 y Leu 5 s Pro s Ala r Arg a Ser 920 r Thr 5 Phe SArg SHis SAla 1000 Ser Ala Phe Val 825 Val Leu Cys Phe Lys 905 Ser Gln Glu Asn Isp 985 %rg Leu Ile Asn 810 Trp Ser Gly Arg Lys 890 Arg Thr Lys Glu Ser 970 Gln Tyr Met Ala Leu Gly Phe Cys 795 Glu Ile Ala Cys Asn 875 Val Ser Cys Val Thr 955 Ala Gly Lys 78 Ph SAl; Se: Va: Ii~ 86( Thi AlE Ser Gly Ser 940 Gly Asp Pro Ala 0 Phe Lys Phe 1 Glu 845 Tyr SIle SAla Ser SPro 925 Phe SArg Gly Pro Ala 1005 Phe Phe Ile 830 Val Phe Glu Arg Leu 910 Gly Gly Tyr Arg Gln 990 Pro Ala Ile 815 Pro Ile Asn 'Glu Ala 895 Cys Leu Ser Ala Ser 975 Lys Thr SLeu Phe 800 Thr Ala Ala Lys Val 880 Thr Gly Thr Gly Thr 960 Gly Cys Lys 750 Asp Glu Val Gly Thr Leu Glu Ser Pro Gly Gly Ser Lys Glu 1010 Glu Glu Thr 025 1015 Arg Pro 1020 Thr Thr Met

Claims (28)

1. An isolated nucleic acid molecule having a nucleic acid sequence that comprises SEQ ID NO: 3; or the complementary strand of SEQ ID NO: 3, wherein the nucleic acid molecule encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output. oo° o

2. An isolated nucleic acid molecule having a nucleic acid sequence that hybridizes under 0. 0high stringency conditions to SEQ ID NO: 1 or its complementary strand; wherein the nucleic acid molecule encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding 00. environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output. o".o "0000* 3. An isolated nucleic acid molecule that comprises a nucleic acid sequence having sufficient identity with SEQ ID NO: 1 or the coding region of SEQ ID NO: 1; wherein *00.0 the nucleic acid molecule encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding S°environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output.

4. An isolated nucleic acid molecule having a nucleic acid sequence that comprises the coding region of SEQ ID NO: 3; or the complementary strand of the coding region of SEQ ID NO: 3, wherein the nucleic acid molecule encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output. An isolated nucleic acid molecule that comprises a nucleic acid sequence that hybridizes under high stringency conditions to the coding region of SEQ ID NO: 1 or its complementary strand; wherein the nucleic acid molecule encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output. oooo S: 6. An isolated nucleic acid molecule that encodes a polypeptide having an amino acid sequence that comprises SEQ ID NO: 4; wherein the polypeptide assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine o. output.

7. An isolated nucleic acid molecule having a nucleic acid sequence that comprises SEQ ID NO: 3; or the complementary strand of SEQ ID NO: 3, wherein the nucleic acid molecule is a RNA molecule and transcribes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output.

8. A probe having a nucleic acid sequence that hybridizes under high stringency conditions to SEQ ID NO: 1 or its complementary strand; wherein the probe comprises a nucleic acid sequence that encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding -36- environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output.

9. A probe having a nucleic acid sequence that hybridizes under high stringency conditions to the coding region or SEQ ID NO: 1 or its complementary strand; wherein the probe comprises a nucleic acid sequence that encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output. o

10. A probe having a nucleic acid sequence that comprises SEQ ID NO: 3; the complement of SEQ ID NO: 3; the coding region of SEQ ID NO: 3; or the complementary strand of the coding region of SEQ ID NO: 3; wherein the nucleic acid molecule encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output.

11. A vector or plasmid that comprises a nucleic acid sequence that comprises SEQ ID NO: 3; the complementary strand of SEQ ID NO: 3, the coding region of SEQ ID NO: 3, or the complement of the coding region of SEQ ID NO: 3.

12. A vector or plasmid that comprises a nucleic acid sequence that encodes SEQ ID NO: 4 and encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output. -37-

13. A vector or plasmid that comprises a nucleic acid sequence that hybridizes under high stringency conditions to SEQ ID NO: 1, the complement of SEQ ID NO: 1, the coding region of SEQ ID NO: 1, or the complement of the coding region of SEQ ID NO: 1; wherein the nucleic acid molecule encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output.

14. A host cell that comprises a nucleic acid sequence that comprises SEQ ID NO: 3; the complementary strand of SEQ ID NO: 3, the coding region of SEQ ID NO: 3, or the 0 complement of the coding region of SEQ ID NO: 3. A host cell that comprises a nucleic acid sequence that encodes SEQ ID NO: 4 and encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output.

16. A host cell that comprises a nucleic acid sequence that hybridizes under high stringency conditions to SEQ ID NO: 1, the complement of SEQ ID NO: 1, the coding region of SEQ ID NO: 1, or the complement of the coding region of SEQ ID NO: 1; wherein the nucleic acid molecule encodes a polypeptide that assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output.

17. An isolated polypeptide molecule having an amino acid sequence that comprises SEQ ID NO: 4 or the coding region of SEQ ID NO: 4, wherein the polypeptide assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in -38- the surrounding environment; or altering water intake, water absorption or urine output.

18. An isolated polypeptide molecule having an amino acid sequence having sufficient identity with SEQ ID NO: 1 or the coding region of SEQ ID NO: 1; wherein the polypeptide assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output. .19. An isolated polypeptide molecule having an amino acid sequence that is encoded by SEQ ID NO: 3, wherein the polypeptide assists fish in or more of the following functions: sensing ion concentrations in the serum or in the surrounding environment; adapting to changing ion concentrations in the serum or in the surrounding environment; or altering water intake, water absorption or urine output. 9 An antibody that specifically binds to the polypeptide of any one of Claims 17 to 19.

21. A method of regulating salinity tolerance in fish comprising modulating the activation of the polyvalent cation-sensing receptor which permits osmoregulation in aquatic species.

22. A method according to Claim 21 wherein the polyvalent cation-sensing receptor is located in fish in selected epithelial cells of the kidney, bladder, heart, intestine or brain.

23. A method according to Claim 21 wherein the polyvalent cation-sensing receptor is encoded by a nucleic acid sequence according to any one of Claims 1 to 7. -39-

24. A method of increasing the salinity tolerance of fish adapted to a fresh water environment comprising increasing the expression of a polyvalent cation-sensing receptor, which permits osmoregulation in aquatic species, in selected epithelial cells ofthe fish. A method according to Claim 24 wherein the polyvalent cation-sensing receptor is encoded by a nucleic acid sequence according to any one of Claims 1 to 7.

26. A method of decreasing the salinity tolerance of fish adapted to a salt water environment comprising decreasing the expression of a polyvalent cation-sensing receptor, which permits osmoregulation in aquatic species, in selected epithelial cells ofthe fish.

27. A method according to Claim 26 wherein the polyvalent cation-sensing receptor is encoded by a nucleic acid sequence according to any one of Claims 1 to 7.

28. A method of screening for agonists and antagonists of a polyvalent cation-sensing receptor protein which permits osmoregulation in aquatic species, said method ~comprising: isolating the flounder urinary, bladder containing a polyvalent e..0 cation-sensing receptor which permits osmoregulation in aquatic species; weighing the isolated bladder to obtain a pre-experiment weight; exposing the isolated bladder to a solution containing a test compound under conditions for a time sufficient for the test compound to agonize or antagonize the polyvalent cation-sensing receptor present in the isolated bladder; and weighing the bladder after the experimental period to obtain a post- experiment weight, wherein the difference of pre and post experiment weights of the bladder are an indication of water reabsorption.

29. An isolated nucleic acid sequence according to any one of Claims 1 to 7, substantially as described herein with reference to the Figures and/or Examples. A nucleic acid probe according to any one of Claims 8 to 10, substantially as described herein with reference to the Figures and/or Examples.

31. A vector according to any one of Claims 11 to 13, substantially as described herein with reference to the Figures and/or Examples. .60:0,

32. A host cell according to any one of Claims 14 to 16, substantially as described herein i with reference to the Figures and/or Examples. *33. An isolated polypeptide according to any one of Claims 17 to 19, substantially as described herein with reference to the Figures and/or Examples.

34. An antibody according to Claim 20, substantially as described herein with reference to the Figures and/or Examples. A method of regulating salinity tolerance in fish according to any one of Claims 21 to "23, substantially as described herein with reference to the Figures and/or Examples.

36. A method of increasing the salinity tolerance of fish according to Claim 24 or Claim substantially as described herein with reference to the Figures and/or Examples.

37. A method of decreasing the salinity tolerance of fish according to Claim 26 or Claim 27, substantially as described herein with reference to the Figures and/or Examples. -41

38. A method of screening for agonists and antagonists of a polyvalent cation-sensing receptor protein according to Claim 28, substantially as described herein with reference to the Figures and/or Examples. DATED this 16th day of September 2002 Brigham and Women's Hospital DAVIES COLLISON CAVE Patent Attorneys for the applicant oo**