Identification of a Corticotropin-Releasing Factor-Binding Protein in the Plasma Membrane of AtT-20 Mouse Pituitary Tumor Cells and Its Regulation by Dexamethasone*

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Abstract
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.

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