Abstract
Calcitonin gene-related peptide (CGRP) is a neuropeptide with diverse biological properties including potent vasodilating activity. Recently, we reported the cloning of complementary DNAs (cDNAs) encoding the human and porcine CGRP receptors which share significant amino acid sequence homology with the human calcitonin receptor, a member of the recently described novel subfamily of G-protein-coupled 7TM receptors. Activation of this family of receptors has been shown to result in an increase in intracellular cAMP accumulation and calcium release. In this study, we demonstrate that HEK-293 cells expressing recombinant CGRP receptors (HEK-293HR or PR) respond to CGRP with increased intracellular calcium release (EC50 = 1.6 nM) in addition to the activation of adenylyl cyclase (EC50 = 1.4 nM). The effect of CGRP on adenylyl cyclase activation and calcium release was inhibited by CGRP (8-37), a CGRP receptor antagonist. Both effects were mediated by cholera toxin-sensitive G-proteins, but these two signal transduction pathways were independent of each other. While cholera toxin pretreatment of HEK-293PR cells resulted in permanent activation of adenylyl cyclase, the same pretreatment resulted in an inhibition of CGRP-mediated [Ca2+]i release. Pertussis toxin was without effect on CGRP-mediated responses. In addition, CGRP-mediated calcium release appears to be due to release from a thapsigargin-sensitive intracellular calcium pool. These results show that the recombinant human as well as porcine CGRP receptor can independently increase both cAMP production and intracellular calcium release when stably expressed in the HEK-293 cell line.
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References
Amara SG, Jones V, Rosenfeld MG, Ong ES, Evans RM: Alternative RNA processing in calcitonin gene expression generates mRNA encoding different polypeptide products. Nature (London) 268: 240–244, 1982
Poyner DR: Calcitonin gene-related peptide: multiple actions, multiple receptors. Pharmac Ther 56: 23–51, 1992
Dennis T, Fournier A, Cadieux F, Pomerleau F, Jolicouer FB, St. Pierre S, Quirion R: hCGRP (8–37), a calcitonin gene-related peptide antagonist revealing CGRP receptor heterogeneity in brain and periphery. J Pharmacol Exp Ther 254: 123–128, 1990
Laufer R, Changeux JP: Calcitonin gene-related peptide and cyclic AMP stimulate phosphoinositide turnover in skeletal muscle cells. J Biol Chem 264: 2683–2689, 1989
Shimekake Y, Nagata K, Ohta S, Kambayashi Y, Teraoka H, Kitamura K, Eto T, Kangawa K, Matsuo H: Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2+ mobilization, in bovine aortic endothelial cells. J Biol Chem 270: 4412–4417, 1995
Bell D, Schluter K, Zhou X, McDermott BJ, Piper HM: Hypertrophic effects of calcitonin gene-related peptide (CGRP) and amylin on adult mammalian ventricular cardiomyocytes. J Mol Cell Cardiol 27: 2433–2443, 1995
Aiyar N, Rand K, Elshourbagy NA, Zeng Z, Adamou JE, Bergsma DJ, Li YA: cDNA encoding the calcitonin gene-related peptide type 1 receptor. J Biol Chem 271: 11325–11329, 1996
Elshourbagy NA, Adamou JE, Swift AM, Disa J, Mao J, Ganguly S, Bergsma DJ, Aiyar N: Molecular cloning and characterization of the porcine calcitonin gene-related peptide receptor. Endocrinology 139: 1678–1683, 1998
Chabre O, Conklin BR, Lin HY, Lodish HF, Wilson E, Ives HE, Catanzariti L, Hemmings BA, Bourne HR: A recombinant calcitonin receptor independently stimulates 3′,5′-cyclic adenosine monophosphate and Ca2+/inositol phosphate signaling pathways. Mol Endocrinol 6: 551–556, 1992
Abou-Samra AB, Juppner H, Force T, Freeman MW, Kong XF, Schipani E, Urena P, Richards J, Bonventre JV, Potts JT Jr, Kronenberg HM, Segre GV: Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: A single receptor stimulates intracellular accumulation of both cAMP and inositol triphosphates and increases intracellular free calcium. Proc Natl Acad Sci USA 89: 2732–2736, 1992
Jelineck LJ, Lok S, Rosenberg GB, Smith RA, Grant FJ, Biggs S, Bensch PA, Kuijper JL, Sheppard PO, Sprecher CA, O'Hara PJ, Foster D, Walker KM, Chen LHJ, McKernan PA, Kindsvogel W: Expression cloning and signaling properties of the rat glucagon receptor. Science 259: 1614–1616, 1993
Wheeler MB, Lu M, Dillon JS, Leng XH, Chen C, Boyd AE III: Functional expression of the rat glucagon-like peptide-1 receptor, evidence for coupling to both adenylyl cyclase and phospholipase-C. Endocrinology 133: 57–62, 1993
Trimple ER, Bruzzone R, Biden TJ, Meehan CJ, Andreu D, Merrifield RB: Secretin stimulates cyclic AMP and inositol triphosphate production in rat pancreatic acinar tissue by two fully independent mechanisms. Proc Natl Acad Sci USA 84: 3146–3150, 1987
Rampelbergh JV, Poloczek P, Francoys I, Delporte C, Winand J, Robberecht P, Waelbroeck M: The pituitary adenylate cyclase activating polypeptide (PACAP I) and VIP (PACAP II VIP1) receptors stimulate inositol phosphate synthesis in transfected CHO cells through interaction with different G proteins. Biochem Biophys Res Commun 1357: 249–255, 1997
Segre GV, Goldring SR: Receptors for secretin, calcitonin, parathyroid hormone (PTH)/PTH-related peptide, vasoactive intestinal peptide, glucagon-like peptide, growth hormone-releasing hormone and glucagon belong to a newly discovered G-protein-linked receptor family. Trends Endocrinol Metab 4: 309–314, 1993
Aiyar N, Griffin E, Nambi P, Edwards RM, Trill JJ, Ellis C, Bergsma DJ: Characterization of the human angiotensin II type I receptor stably expressed in mouse L cells. Mol Cell Biochem 131: 75–86, 1994
Aiyar N, Nambi P, Griffin E, Bhatnagar P, Feuerstein G: Identification and characterization of calcitonin gene-related peptide receptors in porcine renal medullary membranes. Endocrinology 129: 965–969, 1991
Salomon Y, Londos C, Rodbell MA: A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541–548, 1974
Lysko PG, Webb CL, Yue T, Gu J, Feuerstein G: Neuroprotective effects of tetrodotoxin as a Na+ channel modulator and glutamate release inhibitor in cultured rat cerebellar neurons and in gerbil global brain ischemia. Stroke 25: 2476–2482, 1991
Stroop SD, Moore EE: Intracellular calcium increases mediated by a recombinant human calcitonin receptor. J Bone Miner Res 10: 524–532, 1995
Lin CW, Miller TR, Witte DG, Bianchi BR, Stashko M, Manelli MM, Frail DE: Characterization of cloned human dopamine D1 receptor-mediated calcium release in 293 cells. Mol Pharm 47: 131–139, 1995
Macarthur H, Hecker M, Busse R, Vane, JR: Selective inhibition of agonist-induced but not shear stress-dependent release of endothelial autacoids by thapsigargin Br J Pharmacol 108: 100–105, 1993
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Aiyar, N., Disa, J., Stadel, J.M. et al. Calcitonin gene-related peptide receptor independently stimulates 3′,5′-cyclic adenosine monophosphate and Ca2+ signaling pathways. Mol Cell Biochem 197, 179–185 (1999). https://doi.org/10.1023/A:1006962221332
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DOI: https://doi.org/10.1023/A:1006962221332