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 125I-hCGRP-I(1-37) and 160 pM for the antagonist 125I-hCGRP-I(8-37). Unlabeled hCGRP-I and -II and hCGRP-I(8-37) displaced 125I-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, 125I-[C gamma-(4-azidoanilino)Asp3] hCGRP-I, was prepared by carbodiimide coupling of 4-azidoaniline to 125I-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 (Mr) of 60,000, 54,000, and 17,000. Cross-linking of 125I-hCGRP-I and -II and 125I-hCGRP-I(8-37) to soluble CGRP binding sites using disuccinimidyl suberate revealed three specifically labeled binding proteins with the same Mr. The C-terminal fragment 125I-hCGRP-I(8-37), unlike the intact peptide, was, furthermore, cross-linked specifically to a 95,000 Mr 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 Mr components.(ABSTRACT TRUNCATED AT 250 WORDS)
Stangl
D
D
Research Laboratory for Calcium Metabolism, Department of Orthopedic Surgery, University of Zurich, Switzerland.
Born
W
W
Fischer
J A
JA
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000814:Aniline Compounds / Q000378:metabolism; D001391:Azo Compounds / Q000378:metabolism; D015740:Calcitonin Gene-Related Peptide / Q000031:analogs & derivatives / Q000737:chemistry* / Q000302:isolation & purification; D002531:Cerebellum / Q000737:chemistry*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface / Q000737:chemistry; D012995:Solubility; D013388:Succinimides
D000345:Affinity Labels; D000814:Aniline Compounds; D001391:Azo Compounds; D003432:Cross-Linking Reagents; D018003:Receptors, Calcitonin; D011956:Receptors, Cell Surface; D013388:Succinimides; C070827:4-azidoaniline; D015740:Calcitonin Gene-Related Peptide; C019358:disuccinimidyl suberate