The brain corticotropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. Evidence from chemical cross-linking studies.

D E Grigoriadis,E B De Souza

doi:10.1016/s0021-9258(18)38059-1

Abstract

The ligand binding subunits of the corticotropin-releasing factor (CRF) receptors in brain and anterior pituitary of a number of species have been identified by chemical affinity cross-linking using the homobifunctional cross-linking agent disuccinimidyl suberate and 125I-Tyr0-oCRF (ovine CRF). In homogenates of rat, monkey, and human cerebral cortex, 125I-Tyr0-oCRF was covalently incorporated into a protein of Mr = 58,000. Under identical conditions in the anterior pituitary of rat, monkey, cow, and pig, 125I-Tyr0-oCRF was incorporated into a protein of apparent Mr = 75,000. The specificity of the labeling was typical of the CRF binding site since both the cerebral cortex- and pituitary-labeled proteins exhibited the appropriate pharmacological rank order profile characteristic of the CRF receptor (Nle21,Tyr32-oCRF approximately equal to rat/human CRF approximately equal to ovine CRF approximately equal to alpha-helical CRF(6-41) greater than alpha-helical oCRF(9-41) greater than or equal to oCRF(7-41) greater than rat/human CRF(1-20) approximately equal to vasoactive intestinal peptide). In addition to the major labeled proteins, 125I-Tyr0-oCRF was incorporated into higher molecular weight peptides which may represent precursors and into lower molecular weight components which may represent fragments of the major labeled proteins or altered forms of the CRF binding subunit. In summary, these data indicate a heterogeneity between brain and pituitary CRF receptors with the ligand binding subunit of the brain CRF receptor residing on a Mr = 58,000 protein, while in the anterior pituitary, the identical binding subunit resides on a protein of apparent Mr = 75,000.

Highlights

We higher molecular weight peptides which may represent have used '251-Tyro-oCRFand the homobifunctional crossprecursors and into lower molecular weight compo- linking reagent disuccinimidyl suberate (DSS)to characterize nents which may represent fragments of the major the ligand binding subunit of brain andpituitary CRF receplstnrahueebesbieitudyllineigbndiateg,nptwoIdrnnoebteaseinuMninmdb,sirm=naogia5rnr8sya,ul,at0bnteh0urde0nespdipteroioftdfotueartiimthtnaae,sriwyoCnbfdhrRaiitFclhienaertieCenCcRaeRtFpFhhtroeebetreicsnareodnpwgittneioetgr-rhiorbttrohainrtas,dn.cinoWtgwheep, rpporiigettue,piimontarotwrnyhtkhCieceyRh,Fiiansnirotdeficaaheluploimtdowearennirt.niafpivcpaaratiriooeunnst of abrainCRF molecular weight species including pituitary, the identical binding subunit resides on a protein of apparent M, = 75,000
Membranes oCRF, ovine CRF; DSS, disuccinimidylsuberate; SDS-PAGEs, odium were sedimented by centrifugation at 40,000 X g for 10 min at 4 'C, dodecyl sulfate-polyacrylamide gel electrophoresis; EGTA, [ethylene- resuspended in the same buffer, and centrifuged again bis(oxyethylenenitrilo)]tetraacetic acid
In all the tissues examined, the total incorporation of label (0)was less than 10% of the total radioactivity added, and nonspecific binding (e)was defined in the presence of 1FM ratlhuman CRF

Summary

EXPERIMENTAL PROCEDURES

Mmol) was purchased from Du Pont-New England Nuclear (Boston, MA). Unlabeled ovine andrat/human CRF were purchased from. Membranes oCRF, ovine CRF; DSS, disuccinimidylsuberate; SDS-PAGEs, odium were sedimented by centrifugation at 40,000 X g for 10 min at 4 'C, dodecyl sulfate-polyacrylamide gel electrophoresis; EGTA, [ethylene- resuspended in the same buffer (assay buffer), and centrifuged again bis(oxyethylenenitrilo)]tetraacetic acid. Autoradiograms of '251-Tyro-oCRFcross-linked to brain whether it is a degradation product containing the binding membranes,with longerperiodsof exposure, showefadint site for CRF.Alternatively,since some of the smaller peptides labeling of a protein fragment at M , = 20,000-22,000, albeit (comparable M , values of20,000-22,000) migrate with the with a muchlower intensity. In order to resolve the diffuse labeling observed inthe represents a common precursor(s) tothe respective CRF anterior pituitary into specificallylabeled protein compo- binding proteins. It is unlikely, ,that the M , = 58,000

SLICE NUMBER

Findings

ANTERIOR PITUITARY