Galanin receptor in the rat pancreatic beta cell line Rin m 5F. Molecular characterization by chemical cross-linking.
125I-Galanin was cross-linked to receptor in Rin m 5F cell membranes using the bifunctional reagent disuccinimidyl tartarate. Regardless of the presence of reducing agents, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cross-linked galanin-receptor complexes revealed the presence of a radioactive band at Mr 57,000. Excess unlabeled galanin completely inhibited the labeling of the band while other regulatory peptides had no effect. Labeling of the Mr 57,000 complex was abolished by galanin concentration from 10(-9) to 10(-6) M (IC50 = 5 X 10(-9) M). Initial incubation with 125I-galanin in the presence of increasing concentrations of guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) (10(-7) to 10(-4) M) also inhibited the labeling of the Mr 57,000 complex. Moreover, pretreatment of membranes with pertussis toxin before formation of the covalent galanin-receptor complex, dramatically reduced the labeling of the Mr 57,000 species. Covalent Mr 57,000 galanin-receptor complexes solubilized by Triton X-100 bound specifically to wheat germ agglutinin-concanavalin A-, and soybean-coupled Sepharose, supporting the glycoproteic nature of the galanin receptor. Assuming one molecule of 125I-galanin (Mr 3,000) was bound per molecule of protein, these results suggest that the pancreatic galanin receptor is a glycoprotein with a Mr of 54,000 bearing the recognition site for the ligand and which is coupled with a pertussis toxin-sensitive G protein in the plasma membrane.
Amiranoff
B
B
Unité de Recherches sur la Différenciation et la Neuroendocrinologie de Cellules Digestives, Villejuif, France.
Lorinet
A M
AM
Laburthe
M
M
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003902:Detergents; D019004:Galanin; D007340:Insulinoma; D007700:Kinetics; D037102:Lectins; D008970:Molecular Weight; D009479:Neuropeptides / Q000378:metabolism; D017830:Octoxynol; D010190:Pancreatic Neoplasms; D010455:Peptides / Q000378:metabolism; D011092:Polyethylene Glycols; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism*
D003432:Cross-Linking Reagents; D003902:Detergents; D037102:Lectins; D009479:Neuropeptides; D010455:Peptides; D044088:Receptors, Galanin; D011964:Receptors, Gastrointestinal Hormone; D011092:Polyethylene Glycols; D019004:Galanin; D017830:Octoxynol