Molecular cloning and pharmacological characterization of bovine calcitonin receptor-like receptor from bovine aortic endothelial cells.
A complementary DNA encoding calcitonin receptor-like receptor (CRLR) was isolated from a bovine aortic endothelial cell library. The bovine CRLR has 462 amino acids and 92% homology with the human CRLR. In a reverse transcriptase-polymerase chain reaction assay, bovine CRLR was found to be widely distributed, including in the heart and lungs. Stable transfection of bovine CRLR in human embryonic kidney cells (HEK-293) resulted in specific high-affinity [125I] rat adrenomedulin (rADM)-binding (dissociation constant=145+/-15 pM). ADM-stimulated adenylyl cyclase activity with an EC50 value of 5.0+/-1.2 nM. The human ADM receptor antagonist hADM(22-52) inhibited [125I]rADM-binding and ADM-stimulated adenylyl cyclase activity. Interactions between bovine CRLR and individual receptor activity modifying proteins (RAMPs) were also investigated. Transient co-transfection of bovine CRLR cDNA with human receptor activity modifying protein 1 (hRAMP1) cDNA in HEK-293 cells resulted in the expression of a CRLR that displayed high-affinity binding to calcitonin gene-related peptide. Co-transfection of bovine CRLR with human RAMP2 or RAMP3 cDNAs in HEK-293 cells displayed high-affinity ADM receptors. These observations suggest that in the absence of exogenous RAMPs heterologous expression of bovine CRLR results in an ADM receptor phenotype.
Aiyar
Nambi
N
Department of Cardiovascular Pharmacology, GlaxoSmithKline, UW2510, 709 Swedeland Road, Box 1539, King of Prussia, PA 19406-0939, USA. nambi_aiyar-1@gsk.com
Disa
Jyoti
J
Ao
Zhaohui
Z
Xu
Dong
D
Surya
Arjun
A
Pillarisetti
Kodandaram
K
Parameswaran
Narayanan
N
Gupta
Shalley K
SK
Douglas
Stephen A
SA
Nambi
Ponnal
P
D053607:Adrenomedullin; D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000166:cytology; D058286:Calcitonin Receptor-Like Protein; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin / Q000235:genetics* / Q000378:metabolism; D018000:Receptors, Peptide / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; C545580:Calcrl protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; C545509:Ramp2 protein, rat; C545510:Ramp3 protein, rat; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin; D018000:Receptors, Peptide; D053607:Adrenomedullin