Endocrinology, Vol 132, 744-750, Copyright © 1993 by Endocrine Society

ARTICLES

Photoaffinity labeling of rat calcitonin gene-related peptide receptors and adenylate cyclase activation: identification of receptor subtypes

D Stangl, R Muff, C Schmolck and JA Fischer
Department of Orthopedic Surgery and Medicine, University of Zurich, Switzerland.

Different biological effects of calcitonin gene-related peptide (CGRP) analogs have suggested receptor subtypes. Here we have investigated molecular forms of rat CGRP receptors, ligand binding, and activation of adenylate cyclase. A single class of [125I]alpha-human (h)CGRP binding sites was identified in rat cerebellum, liver, and spleen, with dissociation constants of 206 +/- 70 pM, 128 +/- 23 pM, and 229 +/- 64 pM (mean +/- SEM), respectively. Competition experiments showed the same rank order of displacement of [125I]alpha-hCGRP binding in all the tissues examined with rat alpha-CGRP approximately alpha-hCGRP approximately beta-hCGRP > alpha-hCGRP(8-37) > [acetamidomethyl- Cys2,7]alpha-hCGRP > human amylin > salmon calcitonin. Photoaffinity labeling of CGRP receptors using [125I][C gamma-(4- azidoanilino)Asp3]alpha-hCGRP revealed specifically labeled 71- kilodalton (kDa) binding proteins in the cerebellum, brainstem, and spinal cord, of 74 kDa and 68 kDa in the liver, and of 75-90 kDa in the spleen. Enzymatic N-deglycosylation converted the labeled binding proteins into a common 48-kDa form (44 kDa with the molecular mass of the photoligand subtracted). In the presence of 100 microM guanosine-5'- O-(3-thiotriphosphate), the dissociation constant of [125I]alpha-hCGRP binding remained unchanged in the cerebellum but was increased 3-fold in the liver and spleen, suggesting interaction with GTP-binding proteins. In accordance with these results, adenylate cyclase was stimulated by CGRP in the liver and spleen, but not in the cerebellum and brainstem. Furthermore, the linear analog [acetamidomethyl- Cys2,7]alpha-hCGRP enhanced cAMP formation in the liver but not in the spleen. In conclusion, rat CGRP receptors with tissue-specific N- glycosylation but indistinguishable protein molecular mass have been identified in the cerebellum, brainstem, spinal cord, liver, and spleen. Activation of adenylate cyclase by CGRP in the liver and spleen, but not in the central nervous system, and by the linear analog [acetamidomethyl-Cys2,7]alpha-hCGRP in the liver alone provide evidence for CGRP receptor subtypes.

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