PURIFICATION OF THE GROWTH-HORMONE RELEASING HORMONE-RECEPTOR WITH A C-TERMINAL, BIOTINYLATED AFFINITY LIGAND

The receptor for growth hormone-releasing hormone (GHRH) has been puri fied from bovine pituitary tissue and HEK293 cells transfected with hu man or porcine receptor using a retrievable biotinylated GHRH analog. Custom synthesized [His(1), Nle(27), Biotin-Lys(41)]-human GHRH-(1-41) -NH2 (GHRH(b)) bound to pituitary membranes with affinity comparable t o human GHRH. GHRH(b) which has the biotinyl group on the C-terminus o f the peptide allowed simultaneous binding to both the receptor and st reptavidin agarose. This analog was used directly in the purification of the receptor from pituitary tissue or was modified by incorporation of the photoaffinity group ANBNOS (GHRH(lambda b)), radioiodinated an d used to demonstrate purification of the GHRH receptor from transfect ed HEK293 cell membranes. Membranes were prepared and prebound with th e respective ligand followed by CHAPS-solubilization and application o f the solubilized complex to a strepavidin agarose column. Analysis of eluates from the pituitary tissue purification by silver stained SDS PAGE or of autoradiographs of gels from HEK293 eluates revealed specif ic bands of 52 and 55 kDa, respectively. The higher size of the latter band is expected for the ligand-crosslinked receptor. Both bands disp layed similar mobility shifts of 10 kDa upon treatment with N-glycosid ase, a method previously used to characterize this receptor (1). A 45 kDa band corresponding to the size of the G(S alpha) subunit was also detected in eluates of the silver stained gels, suggesting that the GH RH receptor was retrieved as a heterotrimeric complex. Fold purificati on and yield for this procedure were estimated to be greater than 50,0 00 and 2.6-9%, respectively. (C) 1996 Academic Press, Inc.