Structural Characterization by Affinity Cross-Linking of Glucagon-Like Peptide-1(7–36)Amide Receptor in Rat Brain |
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Authors: | José C Calvo Bernardo Yusta Francisco Mora Enrique Blázquez |
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Institution: | Departamento de Bioquímica y Biología Molecular and; Departamento de Fisiología, Facultad de Medicina, Universidad Complutense, Madrid, Spain |
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Abstract: | Abstract: Specific binding of glucagon-like peptide (GLP)-1(7–36)amide was detected in several rat brain areas, with the highest values being found in hypothalamic nuclei and the nucleus of the solitary tract. In hypothalamus and brainstem homogenate binding of 125I-GLP-1(7–36)amide was time, temperature, and protein content dependent and was inhibited by unlabeled proglucagon-derived peptides. The rank order of potency was GLP-1(7–36)amide ? GLP-1(1–36)amide > GLP-1(1–37) ? GLP-2 > glucagon. Scatchard analysis of the steady-state binding data was consistent with the presence of both high- and low-affinity binding sites in hypothalamus and brainstem. Brain 125I-GLP-1(7–36)amide-binding protein complexes were covalently cross-linked using disuccinimidyl suberate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single radiolabeled band of Mr 56,000 identified in both hypothalamus and brainstem homogenates was unaffected by reducing agents. An excess of unlabeled GLP-1(7–36)amide abolished the band labeling, whereas glucagon had no effect. Other unlabeled GLPs inhibited Mr 56,000 complex labeling with the following order of potency: GLP-1(1–36)amide > GLP-1(1–37) > GLP-2. The binding of 125I-GLP-1(7–36)amide and the intensity of the cross-linked band were similarly inhibited in a dose-response manner by increasing concentrations of unlabeled GLP-1(7–36)amide. Covalent Mr 56,000 125I-GLP-1(7–36)amide-binding protein complexes solubilized by Triton X-100 were adsorbed onto wheat germ agglutinin. Our results suggest that the GLP-1(7–36)amide receptor in rat brain is a glycoprotein with a single binding subunit that has a greater molecular weight but binding features and ligand specificity similar to those of its peripheral tissue counterparts. |
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Keywords: | Rat brain Brain-gut peptides Glucagon-like peptides Affinity cross-linking Glucagon-like peptide-1(7–36)amide receptor |
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