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R. Göke, T. Cole, and J. M. Conlon

ABSTRACT

125I-Labelled glucagon-like peptide-1(7–36)amide was cross-linked to a specific binding protein in plasma membranes prepared from RINm5F rat insulinoma-derived cells using disuccinimidyl suberate. Consistent with the presence of a single class of binding site on the surface of intact cells, only a single radiolabelled band at M r 63 000 was identified by SDS-PAGE after solubilization of the ligand—binding protein complex. The band was not observed when 10 nm glucagon-like peptide-1(7–36)amide was included in the binding assay, but 1 μm concentrations of glucagon-like peptide1(1–36)amide, glucagon-like peptide-2 and glucagon did not decrease the intensity of labelling. No change in the mobility of the band was observed under reducing conditions, suggesting that the binding protein in the receptor is not attached to other subunits via disulphide bonds. In control incubations using plasma membranes from pig intestinal epithelial cells, which do not contain specific binding sites for glucagon-like peptide-1(7–36)amide, no cross-linked ligand-binding protein complex was observed.

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B. Gallwitz, W. E. Schmidt, J. M. Conlon, and W. Creutzfeldt

ABSTRACT

Glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) is a potent stimulator of insulin secretion. Receptors for this hormone have been found on different insulinoma-derived cell lines, e.g. the RINm5F cell line which is derived from a radiation-induced rat insulinoma. To characterize the part of the GLP-1(7–36)amide molecule that is responsible for binding to its receptor on RINm5F cells, binding studies with synthetic C-terminal (GLP-1(21–36)amide) and synthetic N-terminal (GLP-1(7–25)) GLP-1 fragments were carried out. GLP-1(21–36)amide showed dose-dependent binding to the GLP-1(7–36)amide receptor but was approximately 1500 times less potent in inhibiting binding of 125I-labelled GLP-1(7–36)amide than the intact hormone. GLP-1(7–25) at concentrations up to 10 μmol/l did not inhibit binding of label. Neither fragment changed intracellular cyclic AMP concentrations, in contrast to GLP-1(7–36)amide which increased intracellular cyclic AMP. GLP-1(21–36)amide, however, acted as a weak partial antagonist of GLP-1(7–36)amide with respect to GLP-1(7–36)amide-dependent stimulation of cyclic AMP production.