STRUCTURAL-ANALYSIS OF CGRP RECEPTORS ON GASTRIC SMOOTH-MUSCLE AND PANCREATIC ACINAR-CELLS

Calcitonin gene-related peptide (CGRP) immunoreactivity is widely dist ributed in the central nervous system and gastrointestinal (GI) tract, and specific receptors have been described on many GI tissues. In the present study, we compared CGRP receptors on gastric smooth muscle ce lls with those on pancreatic acini from guinea pig with the use of che mical cross-linking techniques combined with various enzymatic digesti ons. I-125-labeled rat CGRP-I demonstrated temperature-dependent satur able binding to both acinar and gastric smooth muscle cell membranes. After binding, membranes were incubated with 1 mM disuccinimidyl suber ate (DSS), solubilized with sodium dodecyl sulfate (SDS), and subjecte d to SDS-polyacrylamide gel electrophoresis. Cross-linked radioactivit y was analyzed by autoradiography. A single broad radioactive band [mo lecular weight (M(r)) 57,000] was seen on cell membranes from both tis sues and after cross-linking to intact cells. These bands were not alt ered by addition of dithiothreitol. This radioactive band was not dete cted without DSS present or with addition of 10 muM rCGRP-I. rCGRP-I i nhibited cross-linking with half-maximal inhibition of 32 nM with memb ranes from both tissues, and there was a close correlation between its ability to inhibit binding and to inhibit cross-linking. Cross-linkin g was not inhibited by non-CGRP related peptides. With membranes from both tissues, N-glycanase digestion increased the mobility of the orig inal band. Neuraminidase digestion only slightly increased the mobilit y of the original band; however, the subsequent addition of O-glycanas e showed no additional effect on both membranes. Endoglycosidase H dig estion had no effect in either tissue. The present results demonstrate that on both tissues the cell membrane receptor for CGRP is an N-link ed sialoglycoprotein. The apparent M(r) of this sialoglycoprotein is 5 7,000, and this polypeptide does not contain disulfide-linked subunits or O-linked carbohydrates.