Human choriogonadotropin-induced coupling of receptor and Gs protein and the effect of hormone deglycosylation.
The detergent-soluble extract of rat ovary plasma membranes contained a Gs protein of about 100 kDa as shown by its elution behavior on a Bio Gel A-1.5m column. However, the cell membranes exposed to hCG (37 degrees C, 15 min) contained in addition a higher molecular weight Gs protein complex of 300 kDa comprised of human chorionic gonadotropin (hCG) receptor (hCGR) and Gs. The complex bound with an affinity column of GTP-Sepharose and could be released with Gpp(NH)p and GTP inhibited this binding. The presence of the hCGR in the complex was shown by its binding to 125I-hCG. Furthermore, GTP inhibited the binding of hCG to the complex. These results indicate the presence of hCGR and Gs protein complex in the hCG-treated membranes. hCGR and Gs protein were individually purified and reconstituted into phospholipid vesicles. The protein-phospholipid vesicles showed saturation kinetics of binding of 125I-hCG and 3H-Gpp(NH)p. Incubation of phospholipid vesicles with hCG resulted in a 2-3-fold increase in the binding of 3H-Gpp(NH)p and GTPase activity. Activation of Gs protein was dependent on the length of incubation and the hormone concentration. Deglycosylated hCG was about 10 times less potent than hCG suggesting a role of carbohydrates of hCG in inducing hCG-Gs protein interactions. The data with the in vitro reconstitution system rule out the involvement of a carbohydrate-binding lectin in the function of the hormone.
Seth
P K
PK
Department of Biological Sciences, SUNY, Buffalo 14260.
Bahl
O P
OP
D000818:Animals; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008081:Liposomes; D015636:Magnesium Chloride / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012969:Sodium Fluoride / Q000494:pharmacology
D006063:Chorionic Gonadotropin; D008081:Liposomes; D010743:Phospholipids; D015636:Magnesium Chloride; D012969:Sodium Fluoride; D019204:GTP-Binding Proteins