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Solubilization of active GLP‐1 (7–36)amide receptors from RINm5F plasma membranes
Author(s) -
Göke Rüdiger,
Oltmer Britta,
Sheikh Søren P.,
Göke Burkhard
Publication year - 1992
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(92)80852-8
Subject(s) - receptor , chaps , chemistry , gtp' , membrane , biochemistry , peptide , amide , binding site , g protein coupled receptor , enzyme
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‐β‐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 conentration‐dependently by addition of unlabeled peptide. Scatchard analysis of binding data revealed a single class of binding sites with K a = 0.26 ± 0.03 nM and B max = 65.4 ± 21.24 fmol/mg of protein for the membrane‐bound receptor and K a = 22.54 ± 4.42 μM and B max = 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‐γ‐S nor GDP‐β‐S affected binding or 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.