Solubilization of active GLP-1 (7-36)amide receptors from RINm5F plasma membranes.
Glucagon-like peptide-1 (7-36)amide (GLP-1 (7-36)amide) represents a physiologically important incretin in mammals including man. Receptors for GLP-1 (7-36)amide have been described in RINm5F cells. We have solubilized active GLP-1(7-36)amide receptors from RINm5F cell membranes utilizing the detergents octyl-beta-glucoside and CHAPS; Triton X-100 and Lubrol PX were ineffective. Binding of radiolabeled GLP-1(7-36)amide to the solubilized receptor was inhibited concentration-dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with Kd = 0.26 +/- 0.03 nM and Bmax = 65.4 +/- 21.24 fmol/mg of protein for the membrane-bound receptor and Kd = 22.54 +/- 4.42 microM and Bmax = 3.9 +/- 0.79 pmol/mg of protein for the solubilized receptor. The binding of the radiolabel to the solubilized receptor was dependent both on the concentrations of mono- and divalent cations and the protein/detergent ratio in the incubation buffer. The membrane bound receptor is sensitive to guanine-nucleotides, however neither GTP-gamma-S nor GDP-beta-S affected binding of labeled peptide to solubilized receptor. These data indicate that the solubilized receptor may have lost association with its G-protein. In conclusion, the here presented protocol allows solubilization of the GLP-1(7-36)amide receptor in a functional state, and opens up the possibility for further molecular characterization of the receptor protein.
Göke
R
R
Department of Internal Medicine, Phillipps-University of Marburg, Germany.
Oltmer
B
B
Sheikh
S P
SP
Göke
B
B
D000818:Animals; D002412:Cations / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D005934:Glucagon / Q000378:metabolism*; D052216:Glucagon-Like Peptide 1; D000067757:Glucagon-Like Peptide-1 Receptor; D004763:Glucagon-Like Peptides; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D007340:Insulinoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D010455:Peptides / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism; D018027:Receptors, Glucagon; D012995:Solubility; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002412:Cations; D003902:Detergents; C000593835:Glp1r protein, rat; D000067757:Glucagon-Like Peptide-1 Receptor; D010446:Peptide Fragments; D010455:Peptides; D011956:Receptors, Cell Surface; D018027:Receptors, Glucagon; D013873:Thionucleotides; C061970:glucagon-like peptide 1 (7-36); D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004763:Glucagon-Like Peptides; C023427:guanosine 5'-O-(2-thiodiphosphate); D052216:Glucagon-Like Peptide 1; D005934:Glucagon