Identification and characterization of a pituitary corticotropin-releasing factor binding protein by chemical cross-linking

A corticotropin-releasing factor (CRF) binding protein has been identified based on the chemical cross-linking of ovine Nle21,m-125I-Tyr32]CRF (125I-oCRF) to bovine anterior pituitary membranes using disuccinimidyl suberate (DSS). The apparent molecular weight of the cross-linked complex determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography was approximately 75,000 and was slightly decreased in its nonreduced state, suggesting the presence of intramolecular disulfide bonds. Subtracting the molecular weight of 125I-oCRF, the binding protein appeared to have a molecular weight of approximately 70,000. The cross-linking was specific since an excess (1 microM) of an unrelated peptide (insulin) did not affect the appearance of the Mr 75,000 band. The concentration of CRF required to inhibit cross-linking by 50% was found to be similar to that determined for bovine pituitary CRF receptors by radioreceptor assay. The nonhydrolyzable GTP analogue 5'-guanylylimidodiphosphate dose dependently inhibited the cross-linking of 125I-oCRF to the Mr 70,000 protein. 50 nM of the inactive CRF analogue, Ala14]oCRF, had no effect on the cross-linking, an observation which is consistent with this compound's low potencies in bioassays and radioreceptor assays. These results strongly suggest that this Mr 70,000 protein is the biological bovine anterior pituitary CRF receptor.