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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.

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B Gallwitz, M Witt, U R Fölsch, W Creutzfeldt, and W E Schmidt

ABSTRACT

Glucagon-like peptide-1(7–36)amide (GLP-1(7–36) amide) and gastric inhibitory polypeptide (GIP), peptides of the glucagon family, stimulate insulin secretion in vitro and in vivo. They possess high N-terminal sequence homology. Binding studies with 125I-labelled GIP and 125I-labelled GLP-1(7– 36)amide were performed in RINm5F insulinoma cells to investigate receptor specificity and to compare both receptors directly. Both binding sites were highly ligand-specific: GIP did not bind to the GLP-1(7–36)amide receptor and vice versa. Both peptides increased intracellular cyclic AMP levels; GLP-1(7– 36)amide was 100-fold more potent in stimulating cyclic AMP production when compared with GIP. At ranges of 1–10 nmol GLP-1(7–36)amide/1 and 0·1–10 GIP/1, corresponding to submaximal binding concentrations, the hormones showed an additive effect on cyclic AMP production. The N-terminal portion of GIP was important for binding, as GIP(1–30) showed almost full binding and biological activity. GIP(17–42) bound in a concentration-dependent manner with approximately 500-fold lower potency than GIP. At concentrations of up to 10 μmol GIP(17–42)/1 no stimulation of cyclic AMP was observed.

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R Walli, H Schäfer, C Morys-Wortmann, G Paetzold, R Nustede, and W E Schmidt

ABSTRACT

Human galanin (hGal) is an important neuro-modulator present in the brain, gastrointestinal system and the hypothalamo-pituitary axis. A specific receptor for hGal has been identified in various areas in human brain. A single class of high affinity binding sites was found on plasma membranes of the amygdala (K d 0·23 nm, Bmax 44 fmol/mg), the hypothalamus (K d 0·20 nm, Bmax 25 fmol/mg) and the cortex cerebri (K d0·11 nm, Bmax 8·2 fmol/mg). Other brain areas, i.e. cerebellum, thalamus or pons, expressed binding sites of identical high affinity in lower quantities (Bmax <3 fmol/mg). Specific binding of 125I-labelled hGal was found to be reversible, time- and temperature-dependent and inhibited by Ca2+, Na+ and K+ ions at a concentration of 5 mm. Non-hydrolysable guanosine nucleotides potently reduced specific binding of 125I-labelled hGal by more than 80%. Synthetic hGal analogues substituted in the N-terminal region exhibited strongly reduced binding affinity for the hGal receptor. Using 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulphonate, hGal receptors were successfully solubilized from human cortical membranes, exhibiting no significant loss of binding affinity. Affinity cross-linking to 125I-labelled hGal revealed a labelled band of approximately 60 kDa sensitive to unlabelled Gal. This putative hGal receptor is glycosylated since its molecular size was reduced after treatment with endoglycosidase F. Receptors bound to 125I-labelled hGal could be specifically adsorbed to wheat germ agglutinin and ricinus communis agglutinin, suggesting that receptor glycosylation involves N-acetyl glucosamine and galactose respectively.