CHARACTERIZATION AND PHOTOAFFINITY-LABELING OF A CALCITONIN GENE-RELATED PEPTIDE RECEPTOR SOLUBILIZED FROM HUMAN CEREBELLUM

Calcitonin gene-related peptide (CGRP) receptors were solubilized from human (h) cerebellum with use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of CGRP binding sites with apparent dissociation constants of 50 pM for the intact I-125-hCGRP-I(1-37) and 160 pM for the antagonist I-125-hCGRP-I(8-37). Unlabeled hCGRP-I and -II and hCGRP-I(8-37) displaced I-125-hCGRP-I from solubilized CGRP receptors with similar potencies (ID50 = 70-150 pM). Human CGRP-I(15-37), -(21-37), and -(28-37) were less potent (ID50 greater-than-or-equal-to 70 nM), suggesting that amino acid residues 8-14 may be important for maintaining high binding affinity. A novel photoreactive analogue of hCGRP-I, I-125-[C-gamma-(4-azidoanilino)Asp3]hCGRP-I, was prepared by carbodiimide coupling of 4-azidoaniline to I-125-hCGRP-I. Photoaffinity labeling of soluble CGRP receptors with the photoreactive analogue and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed three specifically labeled binding proteins with apparent molecular weights (M(r)) of 60 000, 54 000, and 17 000. Cross-linking of I-125-hCGRP-I and -II and I-125-hCGRP-I(8-37) to soluble CGRP binding sites using disuccinimidyl suberate revealed three specifically labeled binding proteins with the same M(r). The C-terminal fragment I-125-hCGRP-I(8-37), unlike the intact peptide, was, furthermore, cross-linked specifically to a 95 000 M(r) protein. The CGRP receptor is N-glycosylated. Treatment with endoglycosidase F/N-glycosidase F converted the 60 000 and 54 000 to 46 000 and 41 000 M(r) components. In conclusion, binding and labeling data indicate that the N-terminal ring structure of CGRP is not essential for the interaction with the receptor from the human cerebellum. Moreover, side-chain modification of Asp3 of hCGRP-I does not interfere with receptor binding.