Significance of the carbohydrate moiety of the rat ovarian luteinizing-hormone/chorionic-gonadotropin receptor for ligand-binding specificity and signal transduction.
The contribution of the carbohydrate moiety of the rat ovarian luteinizing-hormone (LH)/chorionic-gonadotropin (CG) receptor to ligand-binding specificity and signal transduction was investigated by using glycosidases. Purified membranes from pseudo-pregnant rat ovaries were treated with neuraminidase or peptide N-glycosidase F, to remove terminal sialic acids and N-linked oligosaccharides of the receptor, respectively. Ligand blotting and densitometric scanning of the autoradiograms showed that 90-95% of the receptors were deglycosylated, and that desialylation was virtually complete. Neither the desialylated nor the deglycosylated receptors were able to bind human follicle-stimulating hormone or bovine thyroid-stimulating hormone, as revealed by competition binding experiments. The 50% effective dose of hCG for adenylate cyclase activation, as determined by measuring the formation of cyclic [32P]AMP from [alpha-32P]ATP for 15 min at 30 degrees C, was similar in the control and deglycosylated membranes: 10.2 +/- 3.3 nM and 12.2 +/- 3.8 nM respectively. The same was true for the time course of the basal, hCG- and forskolin-stimulated enzyme activity. In addition, removal of oligosaccharides from the receptor did not restore the ability of desialylated hCG, nor of the deglycosylated hormone, to stimulate adenylate cyclase. In conclusion, the carbohydrate moiety of the native membrane-inserted rat ovarian LH/CG receptor does not contribute to the ligand-binding specificity, and it is not required for the functional coupling of the occupied receptor and the adenylate cyclase system. These functions are associated with the polypeptide portion of the receptor.
Petäjä-Repo
U E
UE
Biocenter, University of Oulu, Finland.
Merz
W E
WE
Rajaniemi
H J
HJ
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000378:metabolism / Q000494:pharmacology*; D004789:Enzyme Activation; D005260:Female; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D010053:Ovary / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011974:Receptors, LH / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity
D006063:Chorionic Gonadotropin; D008024:Ligands; D008562:Membrane Glycoproteins; D011974:Receptors, LH; D005640:Follicle Stimulating Hormone; D000262:Adenylyl Cyclases