Receptor-linked proteolysis of membrane-bound glucagon yields a membrane-associated hormone fragment.
We have studied, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, glucagon-receptor complexes that arise from the incubation of canine hepatic plasma membranes with [[125I]iodo-Tyr10]glucagon. While a 54,000-dalton membrane protein was tentatively identified as the glucagon receptor by chemical cross-linking, an additional component having an apparent molecular weight of 30,000 was routinely identified as also resulting from glucagon-receptor interactions. The latter material, however, was not observed when gels were fixed prior to autoradiography and was not affected by the addition of cross-linking agents to membrane incubations. Subsequent analysis actually identified the material as a fragment of radiolabeled glucagon that contains at least residues 1-13, has no ability of its own to associate with plasma membranes, and remains tightly membrane bound once it has been formed by receptor-mediated processes. Formation of the fragment was inhibited by phenylmethylsulfonyl fluoride, glucagon, and GTP, but not by N-ethylmaleimide or by a variety of glucagon-related peptides. Overall, our results identify a proteolytic modification of glucagon this is linked to the binding of ligand to high affinity GTP-dependent receptors and the existence of a physically distinct state of receptor in which the binding site is tightly filled by a ligand fragment.
Sheetz
M J
MJ
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Tager
H S
HS
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005934:Glucagon / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018027:Receptors, Glucagon
D010446:Peptide Fragments; D011480:Protease Inhibitors; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D006160:Guanosine Triphosphate; D005934:Glucagon