Covalent cross-linking of growth hormone-releasing factor to pituitary receptors.
We have used a bifunctional cross-linker, disuccinimidyl suberate, to covalently attach [125I]human pancreatic GH-releasing factor (GHRF) (-1-40)OH to bovine pituitary membranes and rat anterior pituitary cells. Covalently radiolabeled membrane and cell preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. In the former case, we observed the specific labeling of a polypeptide with an apparent mol wt of 75,000 +/- 3,000. The labeling of this species was specific for GHRF, as evidenced by the fact that it was inhibited in a dose-dependent fashion with increasing concentration of unlabeled GHRF. Furthermore, the radiolabeling was inhibited in the presence of excess unlabeled GHRF analogs but not unrelated peptides such as insulin and rat GH. The size of the radiolabeled band was the same in both bovine pituitary membranes and rat anterior pituitary cells. The extent of radiolabeling was dependent on the amount of membrane or the number of cells present during the binding reaction. These observations indicate that the mol wt 75,000 species is a ligand-binding subunit of the GHRF receptor in the pituitary. Under nonreducing conditions, a species much larger than mol wt 200,000 was specifically radiolabeled, again in both bovine pituitary membranes and rat cells. This result suggests the possibility that the ligand-binding subunit might be disulfide-linked to other subunit(s) forming homo- and heterooligomers.
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D013007:Growth Hormone-Releasing Hormone / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D013388:Succinimides / Q000494:pharmacology
D000345:Affinity Labels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D013388:Succinimides; C036893:somatotropin releasing factor 40; D013007:Growth Hormone-Releasing Hormone; C048250:somatotropin releasing hormone receptor; C019358:disuccinimidyl suberate