Photoaffinity cross-linking to the pituitary receptor for growth hormone-releasing factor.
Photoaffinity cross-linking methods presented here demonstrate a 55-kilodalton (kDa) GH-releasing factor (GRF) receptor in ovine pituitary membranes and in cell lines expressing the cloned human pituitary receptor complementary DNA. Covalent cross-linking of photoprobe to this high affinity site is strongly competed by 1 nM GRF. Competition shows strong specificity for GRF over related peptides. Reduced cross-linking in the presence of guanosine 5'-O-(3-thiotriphosphate) suggests that this is a G-protein-coupled receptor. Detection of cross-linking to this receptor required detergent extraction to reduce high nonspecific binding of GRF photoprobe. Partial deglycosylation of the cross-linked receptor with neuraminidase caused a shift in apparent size to 52 kDa. Complete deglycosylation with N-glycosidase caused a shift to 45 kDa, demonstrating that this receptor is an N-linked glycoprotein and agreeing with the protein size and single glycosylation site predicted from the cloned complementary DNA sequence. These sizes differ from those found in previous reports which used chemical cross-linking to identify GRF receptor. This photoaffinity cross-linking method will facilitate studies of receptor function and tissue distribution. Photoaffinity cross-linking can also be used to map regions of the receptor molecule and bound GRF that are in close proximity.
Gaylinn
B D
BD
Department of Medicine, University of Virginia School of Medicine, Charlottesville 22908.
Lyons
C E
CE
Zysk
J R
JR
Clarke
I J
IJ
Thorner
M O
MO
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002793:Cholic Acids; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D009439:Neuraminidase / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D018013:Receptors, Neuropeptide / Q000378:metabolism*; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000378:metabolism*; D012756:Sheep
D002793:Cholic Acids; D003432:Cross-Linking Reagents; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D006026:Glycoside Hydrolases; D009439:Neuraminidase; C048250:somatotropin releasing hormone receptor; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate