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 purified from bovine pituitary tissue and HEK293 cells transfected with human or porcine receptor using a retrievable biotinylated GHRH analog. Custom synthesized [His1, Nle27, Biotin-Lys41]-human GHRH-(1-41)-NH2 (GHRHb) bound to pituitary membranes with affinity comparable to human GHRH. GHRHb which has the biotinyl group on the C-terminus of the peptide allowed simultaneous binding to both the receptor and streptavidin 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 (GHRHlambdab), radioiodinated and used to demonstrate purification of the GHRH receptor from transfected HEK293 cell membranes. Membranes were prepared and prebound with the respective ligand followed by CHAPS-solubilization and application of the solubilized complex to a streptavidin agarose column. Analysis of eluates from the pituitary tissue purification by silver stained SDS PAGE or of autoradiographs of gels from HEK293 eluates revealed specific bands of 52 and 55 kDa, respectively. The higher size of the latter band is expected for the ligand-crosslinked receptor. Both bands displayed similar mobility shifts of 10 kDa upon treatment with N-glycosidase, a method previously used to characterize this receptor. A 45 kDa band corresponding to the size of the Gs alpha subunit was also detected in eluates of the silver stained gels, suggesting that the GHRH receptor was retrieved as a heterotrimeric complex. Fold purification and yield for this procedure were estimated to be greater than 50,000 and 2.6-9%, respectively.
Zysk
J R
JR
Agricultural Research Division, American Cyanamid Company, Princeton, New Jersey 08543, USA.
Gaylinn
B D
BD
Lyons
C E
CE
Johnson
B
B
Eppler
C M
CM
Baumbach
W R
WR
Thorner
M O
MO
D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002460:Cell Line; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D010902:Pituitary Gland / Q000378:metabolism; D011485:Protein Binding; D018013:Receptors, Neuropeptide / Q000302:isolation & purification* / Q000378:metabolism; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000302:isolation & purification* / Q000378:metabolism; D013552:Swine
D008024:Ligands; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; C048250:somatotropin releasing hormone receptor