SOLUBILIZATION OF ACTIVE RECEPTORS FOR GLUCAGON-LIKE PEPTIDE-1 (7-36)AMIDE FROM RAT LUNG MEMBRANES

We report on a protocol that allows the solubilization of active glucagon-like peptide (GLP)-1-(7-36)amide receptors from rat lung membranes. Digitonin-solubilized GLP-1(7-36)amide binding proteins from lung membranes most effectively, whereas (3-[(3-cholamidopropyl)-dimethylamino]-1-propane-sulfonate was less affective, and octyl-beta-glucoside, Triton X-100 and Lubrol PX were almost ineffective. Solubilization of binding activity was optimal at a digitonin concentration of 1%, a protein-to-detergent ratio of 1:10, and a pH between 7.0 and 8.0. Binding of GLP-1(7-36)-amide to solubilized receptors was dependent on the concentration of solubilized protein. The presence of certain mono- and divalent cations was crucial for binding of GLP-1(7-36)amide to solubilized receptors. Scatchard analysis of the binding data revealed a single class of binding sites with dissociation and maximum binding constant values of 0.40 +/- 0.20 nM and 80.0 +/- 26.0 26.0 fmol/mg protein for membrane bound and 7.0 +/- 0.6 muM and 12.0 +/- 6.0 nmol/mg protein for solubilized receptors, respectively. In cross-linking experiments I-125-labeled GLP-1-(7-36)amide was covalently attached to GLP-1(7-36)amide receptors on lung membranes. The apparent molecular mass of the solubilized receptor was 55,000 Da. This was proven in another experiment when receptor was consecutively cross-linked after solubilization. Nonhydrolyzable GTP analogues (GTPgammaS or GDPbetaS) were unable to reduce GLP-1(7-36)-amide-binding at solubilized receptors. This argues that the receptor is solubilized as a single protein and not as a receptor-G protein complex. A partial purification of the solubilized receptor protein utilizing lectin columns indicated that the GLP-1-(7-36)amide receptor is a glycoprotein.