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

E Nishimura,N Billestrup,M Perrin,W Vale

doi:10.1016/s0021-9258(18)45139-3

Abstract

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.

Highlights

To be initiated by its binding to specific receptors which have been identified in therat [5, 6] and bovine [7] anterior pituitary
Corticotropin-releasing factor (CRF) binding can be affected by divalent cations, as well as tnwltdioeiineiumAnkibmgiicodhhnodvyatgrsoilsonutdoifbebcfetoeechaoertyrnvenalosittcnuieepdrreliofeniaons[-tsrrtNe-i(el-lfDplpiieeneioS2atdkluS'syeiib,)antd.raacgcnsrrToeyy-fmdhal1aaecm2pompt6nloepe1irmdxat-(heCrTbdeeyRrnecramtFgthe3ne)oer2elbmml]se(eiCcn"iilucnRue'dIasceFl-iialotdnnrrCgcgobrRppyodFshrsoi)ooss----ucctbiirinnhey-vgceIenograpluedovtagoerenudnrmy,leyalerlwtannotetoutreyochilcnfapeytvvorhceoetlesitadteusaieigesdnseae(sdt(1ne7a2yt)rt,lh.hea1eTte3ethhb)mec.oiysfouueclgnelfahccistnuteidlotasoinrynacpsglotrseuocmfppruloeresrstithstnih-eeclserienosrGkufeiTcgntehggPpeetasboCtgirntRehdtnFoe-t resis followed by autoradiographywas approximately disuccinimidyi suberate (DSS) to identify and characterize 75,000 and was slightly decreased in its nonreduced CRF binding proteinspresent in bovine anteriorpituitary state, suggestingthe presence of intramolecular disul- membranes
The nonhydrolyzable GTP analogue 5'- DTT and Gpp(NHw)pere from Sigma.DSS was obtained from Pierce guanylylimidodiphosphatedose dependently inhibited Chemical Co.All reagents required for SDS-PAGE were purchased the cross-linking of 1261-oCRF to the M, 70,000 pro- from Bio-Rad as was the protein kit used for protein determinations

Summary

Introduction

To be initiated by its binding to specific receptors which have been identified in therat [5, 6] and bovine [7] anterior pituitary. 50 nM of theinactive CRF analogue, [Ala14]oCRF, Membrane Preparation-Plasma membranes were prepared from hadno effect onthe cross-linking, anobservation bovine pituitaries as previously described [7].Briefly, anterior pituiwhich is consistent with this compound's low potencies in bioassays andradioreceptor assays. Tion of this article were defrayed in part by the payment of page Cross-linking-Ovine [Nle21,Tyr32]CRF(oCRF) was iodinated as charges.

Results

Conclusion