Receptors for calcitonin, calcitonin gene related ...

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Receptors for calcitonin, calcitonin gene related peptide, amylin, and adrenomedulling Roman Muff, Walter Born, and Jan A. Fischer

Abstract: Calcitonin, calcitonin gene related peptide, amylin, and adrenomedullin are structurally related polypeptides characterized by a six or seven amino acid ring structure linked by a disulfide bridge and an amidated C-terminus. They exhibit overlapping biological actions as a result of cross-reactivity between the different receptors. Hn this article, the respective receptors and G-protein-coupled postreceptor events are reviewed in relation to some of the biological actions of the peptides. Key words: adrenomedullin, amylin, calcitonin gene related peptide, cyclic AMP, receptors.

RCsurnC : La calcitonine, le peptide BC i au gBne de la calcitonine, l'amyline et l'adrCnomCdu8line sont des polypeptides de structure voisine, caract6risCs par une structure cyclique de six ou sept acides aminks lies par un pont disulfure et dotes d'une extrkmitk C-terminale amidke. Leurs actions biologiques se chevauchent en raison d'une activitk croiske entre les diffkrents rtcepteurs. Bans Ba prksente Ctude, on examine les rkcepteurs respectifs et les effets post-rdcepteurs coupBCs B la protCine G en fonction de quelques actions biologiques des peptides. Mots c l h : adrCnomkdulline, amyline, peptide li6 au gkne de la calcitonine, AMP cyclique, rtcepteurs. [Traduit par la Rtdaction]

Introduction Cdcitonin cdcitonin gene peptide (CGRP) amylin' and adrenomedullin (ADM) belong to a of structurally and biologically related polypeptides. The amino acid sequences range from 32 to 52 residues (Fig. 1). The polypeptides have in common a six or seven amino acid ring structure linked by a disulfide bridge. Sequence kornolcsgy is highest (92%) between human (h) CGRP-I and CGRP-11, followed by 46 % between CGRP-I1 and mylin. The sequence including the ring structure is essential for agonistic activity. Its removal in CT, CGRP, amylin, and ADM converts the peptides into receptor antagonists (Ckiba et d. 1989; Feyen et al. 1992; Young et al. 1994; Eguchi et al. 1994b). Deletion of the 14 amino acid sequence N-terminal to the ring structure, unique in ADM, does not affect vasodilatory activity (Hao et al. 1994). Much Bike in many neuropeptides, the C-termini are amidated. The free acids of CT, CGRP, amy9

9

Received September 6, 1994.

R. Muff, W. Born, and J.A. Fischer." Research Laboratory for Calcium Metabolism, Department of Orthopedic Surgery and Department of Medicine, University of Zurich, 8W8 Zurich, Switzerland. "This

paper was presented at the satellite symposium of the XHH~1UPPTA.R Congress entitled The Second International Symposium on CGRP, 4 - 5 August 1994, Montreal, Quebec, and has undergone the Journal's usual peer review. Author for correspondence at Klinik Balgrist, Forckstrasse 340, 8W8 Zurich, Switzerland.

lin, and ADM have absent or reduced biological activity (Moran et al. 1978; Roberts et al. 1989; ~ o i i s h i t aet a[. 1990; Eguchi et al 19946). In this article the binding characteristics, distribution of receptors, and coupling to secondmessenger pathways are reviewed.

CP receptors CT is synthesized and secreted from the C-cells of the thyroid gland and acts in an endocrine fashion in bone and kidney and in the periventricular region of the centrd nervous system. In the skeleton, CT inhibits bone resorption through inhibition of osteoclastic activity. In the kidney, CT stimulates the urinary excretion of calcium, sodium, chlsride, and phosphate. In the brain, antinociceptive effects are therapeutically important (for a review see Born and Fischer 1993). CT receptors have been identified in osteoclasts, kidney cortex, and in the central nervous system (Marx et d. 1972; Morel 1983; Fischer et al. 1981). CT receptors linked to adenylyl cyclase have been identified with covalent crosslinking techniques in rat osteoclasts and kidney, and the Mr values are 70 - 85 kDa (Nicholson et d. 1986; Bouizar et al. 1986). Cloning of complementary DNA (cBNA) of CT receptors from a porcine kidney epithelial cell line and from human ovary carcinoma revealed structures of 482 and 490 amino acid residues (Ein et al. 1991 ; Gorn et al . 1992). The receptors belong to the family of G-protein-coupled receptors with seven putative transmembrane domains. There, a subfamily with over 38% amino acid sequence identity includes those

Can. J. Physiol. Phsarmacol. 73: 963 -967 (1995). Printed in Canada / Imprim6 au Canada

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Can. J. Physisl. Pharrnacsl. VoI. 73, 1995

Fig. 1. Amino acid sequences of human ADM, CGWP-1 and -11, arnylin, and

Identical amino acids are indicated by

lines and squares.


vm1t L A H Q

II I Il Rl LI Al Gl L/ I I EI AI N F

T R R L A G L

E '

T

~

R

of parathyroid hormone, secretin, vasointestinal peptide, glucagon-like peptide, and growth hormone releasing hormone. Two isoforms with 478 (Cla) and 515 (Clb) amino acid residues have been cloned from rat brain (Sexton et al. 1993). The longer form contains a 37 amino acid insert in the putative second extrace%%ular domain. Expression of the rat CT receptor isoforms in COS-1 cells revealed somewhat different ligand specificity, e.g., human m d rat CT reduced "2"-labelled salmon CT (sCT) binding at b pM by about 70% of the Cla but not of the Clb isoform. Moreover, binding of iz"Ilabelled sCT was reversible in the Clb isoform but largely irreversible in the Cla isofom (Moussami et al. 1994). Nonetheless, boLh isoforrns activate adenylyl cyclase and phospholipase C in response to sCT. With human CT no increase in cytosolic free calcium was detected at up to 1 pM. Two isoforms of the human CT receptor were found in human ovary cells and human thyroid glands (Gorn et d. 1992; Frendo et al. 1994). The CT receptor (hCTR- 1) from ovary cells has 16 additional amino acids in the first intracellular domain not present in all the other cloned CT receptors, including hCTR-2. With the hCTR- 1 receptor, sCT-stimulated adenylyl cyclase was recognized, but inositol phosphate production was not detected (Nussenzveig et a1. 1994). The CT receptor from porcine kidney was stably transfected into proximal tubule cells from the opossum kidney, which express an endogenous parathyroid hormone receptor. In these cells, CT reduced phosphate uptake, much like parathyroid hormone (Muff et al. 1994). The phosphaturic effect of CT observed in vivo (Ardaillou et al. 1967) is therefore mimicked by the expression of the cloned CT receptor in a renal proximal tubular cell line.

Orphan calcitoaain recegtormllke structures Both in rats and humans novel receptors have been cloned that do not recognize any known ligands (Mjulci et al. 1994; Chang et d. 1993; Fliihmann et al. 4995). The derived amino acid sequence is about 60% homologous to the cloned CT receptors. Northern blot analysis revealed that the receptors are expressed mainly in the heart, lung, and kidney but also in liver, skeletal muscle, and pancreas.

CGRP receptors CGRP is mainly synthesized in the central and peripheral nervous system and is considered as a neuropeptide. Pre-

dominant biological actions of CGRP are in the cardiovascular system, where CGWP has vasodilatory , hypotensive, and positive chronstropic and inotropic actions (for a review see Born and Fischer 1993). CGRP receptors have been localized and characterized by binding studies of tissue homogenates and autoradiography throughout the rat and human nervous system, and in peripheral organs such as the heart, spleen, blood vessels, lung, liver, and kidney (for a review see Born and Fischer 1993). A high density of CGWP binding sites was found in rat cerebellum and spinal cord with Wa, values of 71 and 48 m a before and after deglycosylation, respectively (Stangl et al. 1993). With """-labelled CGRP subtracted, the protein mass is 44 kDa (Table 1). Different glycosylation of this 44-kDa CGRP receptor in the central nervous system and the liver and spleen may be due to CGRP receptor subtypes or tissue-specific glycosylation of the same receptor. A distinct distribution of Isblabelled hCGRP-I and hCGRP-I1 binding sites in the periventricdar region of the human brain as revealed by autoradiography provides evidence of different receptors for the closely related peptides (homology 92%) (Hemake et al. 1987). Detailed sfmctural analysis awaits cloning of CGRP receptor(s) encoding cDNA. In cerebellar membranes, CGRP-stimulated adenylyl cyclase was not observed. However, in cultured neonatal mouse cerebellar neurons CGRP stimulated cyclic AMP (CAMP) accumulation with an ECS0of about 1 nM (Stangl et al. 1993; R. Muff and C . Schmidt, unpublished results). In liver and spleen membranes CGRP stimulated CAMP accumulation. Stimulation of CAMP accumulation by the linear analogue [acetarnidomethyl-Cys2v7]hCGRPin liver but not in spleen membranes provides evidence for distinct CGRBl and CGRQ receptor subtypes according to pharmacological evidence provided by the group of Quirion (Dennis et d. 1990; Stangl et al. 1993). Similarly, actions mediated by the CGRP, subtype, unlike the CGRP2 receptor subtype, are suppressed by the N-terminal truncated CGRP (8 - 37), which lacks the ring structure (Dennis et d. 1990). Stbmu%ation of acetylcholine receptor synthesis by CGRP is mediated by activation of adenylyl cyclase and phospholipase C, indicating that the CGRP receptor or subtypes also couple to pkospholipase C (Laufer and Changeux 1989). hCGRP-I has more potent vasodilatory properties and stimulates renin and aldosterone secretion in normal human subjects more than hCGRB-11, whereas the latter is more efficient in reducing pentagastrin-stimulated gastric output

Muff et al. a b l e 1. Receptors for CT, CGRP, arnylin, and ADM in rats.

Glycosylation

M, (kDa)

Peripheral organs

-I-

Lung

-I-

- 53, 57" -67' -44b -70-$6b -M b -6ab nd - ab

Localization


CT

CGRP

Arnylin

Bone Kidney CNS CNS

Insulinoma ADM

VSMC

-

+

ACA

References

Yesa Yes

Houssarni et al. I994 Stangl et al. 1993; R. Muff and C. Schmidt, unpublished

Yes lad

Yes Yes

Bhogal et al. 8992 Moore et al. 1994 Eguchi et al. 1994

Note: ACA, acknylyl cycIase activation; CNS, central nervous system; nd, not done; VSCM, vascular smooth muscle cells. "Cloned brain receptor isoforms; Mr calculated. bMr estimated after cross-linking and gel electrophoresis, after subtraction of the Zk& of the radioligand.

(Beglinger et al. 1991). Whether this is a result of tissuespecific expression of CGRP subtypes remains to be shown.

Arnylin receptors Amylin is synthesized and secreted together wiph insulin in response to glucose from pancreatic @-cells.Biological actions of amylin include inhibition of glucose-stimulated insulin release, increased hepatic glucose output, and inhibition of insulin-stimulated glucose uptake and glycogen synthesis in the skeletal musculature. Endocrine and autocrine actions of arnylin are probable (for a review see Cooper 1994). Amylin interacts at high concentrations (ID5, 240-fold higher than for CGRP), with a bona fide CGRP receptor characterized in the SK-N-MC cell neuroblastoma line, and also with CT receptors (IDSo200-fold higher than for sCT) in the T47D human breast carcinoma cell line (Muff et al. 1992). Similarly, osteoclastic mobility was suppressed by amylin 800- and 12-fold less potently than with salmon or human CT, respectively (Mam et al. 1993). With a distinct receptor discovered in the nucleus accumbens of the rat, inhibition of Bolton Hunter modified rat B251-labelldamylin binding by amylin, sCT, and CGRP is closely related (Sexton et al. 1988; Beaumont et al. 1993). The results of these studies do not allow a distinction to be made between a receptor of amylin, sCT, and CGRP. Indistinguishable inhibition of insulin-stimulated gluconeogenesis in rat soleus muscle by amylin, sCT, and CGRP is consistent with this mylinsCT -CGRP receptor (Leighton and Cooper 1988; Beaumont et al. 1993). Specific amylin binding sites have been identified in membranes of the rat lung (Bhogal et al. 1992). In that study, rat amylin was 100-fold more potent than rat CGRP in the displacement of u51-labelled amylin binding, and sCT was inactive. The lESI-labelledamylin binding protein had an M, vdue sf 63 W>a (Table 1). Another specific amylin receptor coupled to adenylyl cyclase was characterized in rat insulinoma cells (Moore et al. 1994). 'P"-labelled amylin binding inhibition was obtained wiph 40- and 10-fold lower amounts of rat mylin compared with rat CGRP-I and rat CT, respectively. This receptor appears different from that in rat lung since CT was more potent than CGRP.

Adrenomedullin receptors Human ADM was isolated from pheochromocytoma by its ability to stimulate cAMP in platelets (Kitamura et al. 1993a). ADM mRNA of rat and man and immunoreactive human ADM have been localized in several tissues, such as the adrenal medulla and pheochromocytoma and heart, lung, and kidney (Ichiki et al. 1994; Kitamura et al. 1993b; Sakata et d. 1993). ADM and CGRP exhibit similar vasodilatory effects and, as a result, hypotensive properties (Kitamura et al. 1993a; De Wite et al. 1994; Nuki et d. 1993). But in contrast to CGRP, which has positive chronotropic actions in man (Gennari and Fischer 1985), ADM slightly reduces the heart rate (Ishiyama et d.1993; Perret et d.1993). Hmmunoreactive ADM is elevated in the serum of hypertensive patients, suggesting that it is a circulating hormone (Kitamura et al. 1994). Tissue-specific distribution of ABM receptors has not yet been examined in detail, probably because of a lack of suitable radioligands for ADM. In view of the cross-reactivity between ADM and CGRP receptors, they cannot be differentiated with certainty. To this end, in SK-N-MC neuroblastoma cells with a CGRP receptor, hCGRP-I was only 7- and 50-fold more potent than hADM in displacing 1251-labelled CGRP binding and stimulation of cAMP accumulation (Zimmermann et d. 1995; Entzeroth et al. 1995). CGRP stimulated cAMP accumulation threefold more potently than ADM in rat platelets, and was eightfold more potent in its vasodilatory effects in the mesenteric vascular ~ ; et al. 1993). However, beds (Kitamura et al. 1 9 9 3 ~ Nuki vasodilation of the pulmonary vascular beds of the cat was obtained with fourfold lower amounts of ADM compared with CGRP (De Witt et al. 1994). In rat vascular smooth muscle cells, ADM and CGRP revealed indistinguishable EC50 values for the stimulation of cAMP aecumulation (Ishizaka et al. 1994). Species differences cannot be ruled out. In another report also obtained with rat vascular smooth muscle cells, ADM displaced 12"-labelled ADM binding at concentrations 30-fold lower than CGRP (Eguchi et al. 1994~) (Table 1). Half-maximal stimulation of cAMP occurred at sixfold lower concentrations of ADM than CGRP. This effect was antagonized by the CGRP receptor antagonist

Can. J. Physiol. Pharmacol. Vol. 73, 1995


CGWP(8 - 3'7) and by human ADM(22 -52) (Eguchi et al. 1994b). Cross-linking studies witla 1zS14aklled ADM revealed specifically labelled proteins of 120 and 70 kDa, suppressed by excess amounts of both ADM and CGRP. The results are consistent with an ADM receptor in vascular smooth muscle cells with which CGRP interacts. Alternatively, vascular smooth muscle cells may express both ADM and CGWP receptors (Hirata et al. 1988). S o far, no data are available for the coupling of the ADM receptor to phospholipase C.

Distinct receptors of the homologous peptides CT, CGWP, amylin, and ADM have been characterized in many different tissues of several species. The receptors are related functionally as revealed by cross-reactivity of the pegtides in binding studies, as well as by overlapping biological actions. The C T receptor and subtypes thereof, and orphan CT receptor-like structures have in common seven putative transmembrane domains. The structure of the CGWP, m y l i n , and ADM receptors remains to be identified. Interestingly, the protein mass of the deglycosylated CGWP receptor appears smaller by about 100 m i n o acids than the cloned C T receptor isoforms. So far, no data are available for the protein mass of the receptors for amylin and ADM. Calcitsnin receptors are linked to both the adenylyl cyclase and the phospholipase C pathways (Force et al. 1992). CGRP, m y l i n , and ADM stimulate CAMP accumulation, but there is no clear evidence for G-protein-coupled phospholipase C activation. Molecular cloning of the receptors, in addition to those s f CT, and comparison of their structures should reveal whether these receptors lack a phospholipase C activating domain. CGRP and ADM have in common potent vasodilatory effects, amylin is thought to be linked to the etiology of diabetes type 2, and CT is a primordial inhibitor of bone resorption. However, CGRP, amylim, and C T have all these actions in common, yet at different concentrations. The biological relevance of ADM discovered in 1993 remains to be evaluated in more detail.

This work was supported by the Swiss National Science Foun&tisn grant 32.28297.90 and the Kanton of Zurich.

References Alarn, A.S .M.T., Moonga, B.S., Bevis, P.J.R., Huang , C .L.-H., and Zaidi, M. 1993. Arnylin inhibits bone resorption by a direct effect on the motility of rat osteoclasts. Exp. Physiol. 78: 183- 196. Ardaillou, R., Vuagnat, P., MiHhaud, G.,and Richet, G. 1967. Effets de la thyrocalcitonine sur l'excrbtion rbnale des phosphates, du calcium et des ions H+ chez l'hornme. Nephron, 4: 298-314. Beaumont, K., Kenney , M .A ., Young, A. A,, and Rink, T. J. 1993. High affinity amylin binding sites in rat brain. MoH. Pharmacol. 44:493 -497. Beglinger, C., Born, W., Miinch, R.,Kuptz, A., Gutzwiller, J.-B., Jager , K., and Fischer, B. A. 1991. Distinct hernodynamic and gastric effects of hurnan CGWP I and II in man. Peptides (N.Y.), 12: I347 - 1358 .

Bhogal, R., Smith, D.M., and Bloom, S.R. 1992. Investigation and characterization of binding sites for islet amyloid polypeptide in rat membranes. Endocrinology (Baltimore), 130: 906 -9 13. Born, W., and Fischer, J.A. 1993. Calcitonin gene products: molecular biology, chemistry, and actions. Handb. Exp. Pharmacol. 107: 569 -616. Bouizar, Z., Fouchereau-Peron, M., Taboulet, J., Moukhtar, M.S., and Milkaud, 6. 1986. Purification and characterization of calcitonin receptors in rat kidney membranes by covalent cross-linking techniques. Eur. J. Biochem. 155: 141- 147. Chang, C.-P., Bearse, R.V., 11, O'Connell, S . , and Rosenfeld, M.G. 1993. Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron, 18: 1187 - 1195. Chiba, T., Yamaguchi, A., Yamatani, T., Nakarnura, A., Morishita, T., Inui, T., Fukase, M., Noda, T., and Fujita, T. 1989. Calcitonh gene-related peptide receptor antagonist human CGWP-(8-37). Am. J. Bhysiol. 256: E33 1-E335. Cooper, C.J. S. 1994. Amy lin compared with calcitonin generelated peptide: structure, biology, and relevance to metabolic disease. Endocr . Rev. 15: 163-20 B . Dennis, T., Fournier, A., Cadieux, A., Pomerleau, F., Jolicoeur, F.B., St. Pierre, S., and Qeairion, R. 1998. hCGRP a calcitonin gene-related gegtide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery. B. Pharmacol. Exp. Ther. 254: 123- 128. De Witt, B.J., Cheng, D.Y., Carniniti, G.N., Nossaman, B.D., Coy, D.H., Murphy, W .A., and Kadovvitz, P.J. 1994. Comparison of responses to adrenomedullin and calcitonin generelated peptide in the pulmonary vascular bed of the cat. Eur. J. PhamacoB. 257: 303 -306. Eguchi, S., Hirata, Y., &no, H., Sato, K., Watanabe, Y., Watanak, T.X., Nakajima, K . , Sakakibara, S., and Marumo, F. 1994a. Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells. FEBS Lett. 340: 226-230. Eganchi, S., Hirata, Y., Iwasaki, H., Sato, K., Watanabe, T.X., h i , T., Nakajima, K., Sakakibara, S., and Mammo, F. 1994b. Structure-activity relationship of adrenomedullin, a novel vasdilatory peptide, in cultured rat vascular smooth muscle cells. Endocrinology (Baltimore), 835: 2454 -2458. Entzeroth, M., Doods, H.N., Wieland, H.A., and Wienen, W. 1995. Adrenomedullin mediates vasodilation via CGRP, receptors. Life Sci. 96: PLl9-PL25. Feyen, J.W.M., Cardhaux, F., Gamse, R., Bmns, C., Azria, M., and Trechsel, 8%. 1992. N-terminal truncation of salmon calcitonin leads to calcitonin antagonist. Biochem. Biophys. Res. Commun. 187: $ - 13. Fischer, J.A., Sagar, S.M., and Martin, J.B. 1981. Characterization and regional distribution of calcitonin binding sites in the rat brain. Life Sci. 29: 663 -671. Fliihmann, B., Muff, R.,Hunziker, W., Fischer, J.A., and Born, W. 1995. A human orphan calcitonin receptor-like structure. Biwhem. Biophys. Res. Cornmun. 206: 342 -347. Force, T., Bonventre, J.V., Flannery, M.R., Gorn, A.H., Yamin, M., and Goldring, S.R. 1992. A cloned porcine renal calcitonin receptor couples to adenyIyl cyclase and phospholipase C. Am. J. Physiol. 262: Fl118-F1115. Frendo, J.L., Pichaud, F., DeLage Mourroux, R., Bouizar, Z., Segond, N., Moubtar, M.S., and Jullienne, A. 1994. An isoform of the human calcitonin receptor is expressed in TT cells and in medullary carcinoma of the thyroid. FEBS Lett. 342: 214-216. Gennari, C., and Fischer, J.A. 1985. Cardiovascular action sf calcitonin gene-related peptide in humans. Calcif. Tissue Iwt. 37: 581 -584. Gorn, A.H., Lin, H.Y., Yamin, M., Auron, P.E., Flannery, M.R., Tapp, D.R., Manning, C.A., Lodisk, M.F., Krane,

,-,,,


Muff et al. S. M., and Goldring, S.R. B 992. Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. J. Clin. Invest. 90: 1726- 1735. Hao, Q . , Chang, J.-K., Gharavi, H., Fortenberry, Y., Hyman, A., and Eippton, H. 1994. An adrenomedullin (ADM) fragment retains the systemic vasodilator activity of human ABM. Life Sci. 54: 265 -270. Henke, W.,Sigrist, S., Lang, W,, Schneider, J., and Fischer, J.A. 1987. Comparison of binding sites for the calcitonin generelated peptides 1 and II in man. Brain Res. 410: 4Q4-488. Hirata, Y., Takagi, Y., Takata, S., Fukuda, Y., Yoshinmi, H., and Fu~ita,T. 1988. Calcitonin gene-related peptide receptor in cultured vascular smooth muscle and endothelial cells. Biochem. Biophys. Res. Ccemun. 151: 1113- 1121. Houssami, S., Findlay, B.M., Brady, C.L., Myers, D.E., Martin, T.J., and Sexton, P.M. 1994. Hsoforms of the rat calcitonin receptor: consequences for ligand binding and signal transduction. Endocrinology (Baltimore), 135: 183- 198. Ichiki, Y., Kitamura, K., Kangawa, K., Kawarnoto, M., Matsuo, H., and Eto, T. 1994. Distribution and characterization of immunoreactive adrenomedullin in human tissue and plasma. FEBS Lett. 338: 6- 10. Ishiyama, Y., Kitamura, K., Ichiki, Y., Nakamura, S., Kida, O., Kangawa, K., and Eto, T. 1993. Hernodynamic effects of a novel hypotensive peptide, human adrenomedullin, in rats. Eur . 9. Phamacol. 241: 271-273. Ishizaka, Y., Ishizaka, Y., Tanaka, M., Kitamura, K., Kangawa, K., Minamino, N., Matsuo, H.. and Eto, T. 1994. Adrenomedullin stimulates cyclic AMP formation in rat vascular smooth muscle cells, Biochem. Biophys. Res, C o m u n . 200: 642 -646. Kitamura, K., Kangawa, K., Kawamoto, M., Ichiki, Y., Nakarnura, S., Matsuo, H., and Eto, T. 1993a. Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 192: 553 -560. Kitamura, K., Sakata, J., Kangawa, K., Koljima, M., Matsuo, H., and Eto, T. 1993b. Cloning and characterization of cDNA encoding a precursor for human adrenomedullin. Biochem. Biophys. Res. Commun. 194: 720 -725. Kitamura, K., Hchiki, Y., Tanah, M., Kawamoto, M., Emura, J., Sakakibara, S . , Kangawa, K., Matsuo, H., and Eto, %. 1994. Hrnmunoreactive adrenomedullin in human plasma. FEBS Lett. 341: 288-290. Laufer, R., and Changeux, J .-P. 1989. Calcitonin gene-related pegtide and cyclic AMP stimulate phosphoinositide turnover in skeletal muscle cells. J. Biol. Chem. 264: 2683 -2689. Leighton, B., and Cooper, G.J. S. 1988. Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature (London), 335: 632 - 635. Ein, H.Y., Harris, T.L., Flannery, M.S., Amffo, A., Kaji, E.H., Gorn, A., Kolakewski, L.F., Lodish, H.F., and Goldring, S.R. 1991. Expression cloning of an adenylate cyclase-coupled calcitonin receptor. Science (Washington, D.C.), 254: 10221024. Marx, S.J., Woodard, C.J., and Aurbach, G.D. 1972. Galcitonin receptors of kidney and bone. Science (Washington, D.C.), 178: 999-1001. Moore, C.X., Werich, J.P., and Beaumont, K. 1994. High affinity amylin binding sites in RIP3 m5f cells. Proceedings of the 76th Annual Meeting of the Endocrine Society. Anaheim, Calif., June 15-18, 1994. p. 301. (Abstr.) Moran, J., Hunziker, W., and Fischer, J.A. 1978. Cdcitonin and calcium ionophores: cyclic AMP responses in cells of a human lymphoid line. Proc. Natl. Acrtd. Sci. U.S.A. 75: 3984-3988. Morel, F. 1983. Regulation of kidney functions by hormones: a new approach. Recent Prog. Horm. Wes. 39: 271 -304.

Morishita, T., Yarnaguchi, A., Fujita, T., and Chiba, T. 1990. Activation of adenylate cyelase by islet amyloid polypeptide with COBH-termiml amide via calcitonin gene-related peptide receptors on rat liver plasma membrane. Diabetes, 39: 875 -877. Muff, K., Stangl, B., Born, W., and Fischer, J.A. 1992. Conmparisen of a calcitonin gene-related peptide receptor in a human neuroblastoma cell line (SK-N-MC) and a calcitonin receptor in a human breast carcinoma cell line (T47D). Ann. N.Y. Acad. Sci. 657: 106- 116. Muff, K., Kaufmann, M., Born, W., and Fischer, J.A. 1994. Calcitonin inhibits phosphate uptake in opossum kidney cells stably transfected with a porcine calcitonin receptor. Endocrinology (Baltimore). 134: 1593- 1596. Nicholson, G.C., Moseley, J,M., Sexton, P.M., Mendelsohn, F.A.8,, and Martin, T.J. 1986. Abundant calcitonin receptors in isolated rat osteoclasts. P. Clin. Invest. 78: 355 - 360. NjuK, F., Nicholl, C.G., Howard, A., Mak, J.C. W., Barnes, P. J., Girgis, S.I., and Legon, S. 1993. A new ealcitonin-receptor-like sequence in rat pulmonary blood vessels. Clin. Sci. 85: 385-388. Nuki, C., Kawasaki, H., Kitmura, K., Takenaga, M.. Kangawa, K., Eto, T., and Wada, A. 1993. Vasodilator effect of adrenomedullin and calcitonin germe-related peptide receptors in rat msenteric vascular beds. Biochem. Biophys. Res . C o m m n . 1%:245-251. Nussenzveig, B.R., Thaw, C.N., and Gershengorn, M.C. 1994. Inhibition of inositol phosphate second messenger formation by intracellular loop one of a human calcitonin receptor. J. Biol. Chem. 269: 28 123 - 28 129. Perret, M., Broussard, H.,LeGros, T., Burns, A., Chang, J.-K,, Summer, W., Wyman, A., and Lippton, H. 1993. The effect of adrenomedullin on the isolated heart. Life Sci. 53: PL377PL379. Roberts, A.N., Leighton, B., Todd, J.A., Cockburn, D., Schofield, P.N., Sutton, R., Holt, S., Boyd, Y., Day, A.J., Foot, E.A., Willis, A.C., Reid, K.B.M., and Cooper, G.J.S. 1989. Molecular and fitnctional characterization of ainylin, a peptide associated with type 2 diabetes rnellitus. Proc. Natl. Acad. Sci. U.S,A. 86: 9662-9666. Sakata, J., Shimokuh, T., Kitamura, K., Nakarnura, S., Kangawa, K., Matsuo, H.,and Eto, T. 1993. Molecular cloning and bi~lesgicalactivities of rat adrenomedullin, a hypotensive peptide. Biochem, Biophys. Res. Commun. 195: 921 -927. Sexton, P.M., McKenzie, J.S., and Mendelsohn, F.A.O. 1988. Evidence for a new subclass of calcitonin/calcitonin gene-related peptide binding site in rat brain. Neurochem. Int. 12: 323 -335. Sexton, P.M., Houssami, S., Hiltom, J.M., B'Keeffe, E.M., Center, R.J., Gillespie, M.T., Barcy, P., and Findlay, B.M. 1993. Identification of brain isoforms of the rat calcitonin receptor. Mol. Endocrinol. 7: 815 -821. Stangl, D., Muff, W., Schmolck, C., and Fischer, J.A. 1993. Photoaffinity labeling of rat calcitonin gene-related peptide receptors and adenylate cyclase activation: identification of receptor subtypes. Endocrinology (Baltimore), 132: 744 -758. Young, A.A., Gedulin, B., Gaeta, L.S.E., Prickett, K.S.. Beaumont, K., Larson, E., and Rink, T.J. 1994. Selective amylin antagonist suppresses rise in plasma lactate a f er intravenous glucose in the rat-evidence for a metabolic role of endogenous amylin. FEBS Lett. 343: 237-241. Zimmermann, U., Fischer, J.A., and Muff, 8 . 1995. Adrenomedullin and calcitonin gene-related peptide interact with the same receptor in cultured human neuroblastoma SK-N-MC cells. Peptides (N.Y.). In press.