Identification of a corticotropin-releasing factor-binding protein in the plasma membrane of AtT-20 mouse pituitary tumor cells and its regulation by dexamethasone.
CRF stimulates the synthesis and secretion of proopiomelanocortin-derived peptides from AtT-20 mouse pituitary tumor cells. This study has shown that there is a specific binding site for CRF located on the plasma membrane of these cells. Both [125I]iodo-Tyr0CRF and noniodinated CRF (10(-11)-10(-7) M) stimulated, in a dose-dependent manner, the secretion of equimolar amounts of beta-endorphin-like immunoactivity from AtT-20 cells. Disuccinimidyl suberate, a cross-linking agent, was used to demonstrate specific binding of [125I]iodo-Tyr0CRF to plasma membranes from these cells. After cross-linking [125I] iodo-Tyr0CRF, the membrane proteins were solubilized with sodium dodecyl sulfate and electrophoresed on a 10% polyacrylamide gel. A single radioactively labeled band, corresponding to a mol wt of 66,000, was identified by autoradiography. [125I]Iodo-Tyr0CRF binding to these membranes was inhibited by 10(-7) M unlabeled CRF or an equimolar concentration of the CRF analog sauvagine. Similar concentrations (10(-7) M) of TRH, GnRH, insulin, [Arg8]vasopressin, somatostatin, and ACTH did not inhibit [125I]iodo-Tyr0CRF binding to the plasma membranes. Incubation of AtT-20 cells for 24 h in the presence of 10 nM dexamethasone reduced [125I]iodo-Tyr0CRF binding by 80% compared to that in untreated cells. Dexamethasone also inhibited the CRF-stimulated beta-endorphin-like immunoactivity secretory response. These data indicate that binding of CRF to a specific membrane protein is an integral component in the stimulation of AtT-20 cells by CRF.
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D003907:Dexamethasone / Q000494:pharmacology*; D004723:Endorphins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D010902:Pituitary Gland / Q000502:physiology*; D010911:Pituitary Neoplasms / Q000473:pathology; D018019:Receptors, Corticotropin-Releasing Hormone; D017981:Receptors, Neurotransmitter / Q000502:physiology*; D013329:Structure-Activity Relationship; D001615:beta-Endorphin
D003432:Cross-Linking Reagents; D004723:Endorphins; D008565:Membrane Proteins; D018019:Receptors, Corticotropin-Releasing Hormone; D017981:Receptors, Neurotransmitter; D001615:beta-Endorphin; D003907:Dexamethasone