Site specificity of the chorionic gonadotropin N-linked oligosaccharides in signal transduction.
The role of the human chorionic gonadotropin (hCG) N-linked oligosaccharides in receptor binding and signal transduction was analyzed using site-directed mutagenesis and transfection studies. hCG derivatives with alterations at individual glycosylation sites were expressed in Chinese hamster ovary cells. Receptor binding studies showed that absence of any or all of the hCG N-linked oligosaccharides had only a minor effect on the receptor affinity of the derivatives. Similarly, absence of the N-linked oligosaccharides from the beta subunit or a single oligosaccharide from Asn-78 of alpha had no effect on the production of cAMP or on steroidogenesis. However, the absence of carbohydrate at Asn-52 of alpha decreases both the steroidogenic and cAMP responses. Furthermore, absence of this critical oligosaccharide unit on alpha unmasks differences in the two N-linked oligosaccharides on beta; the beta Asn-13 oligosaccharide but not the beta Asn-30 oligosaccharide plays a more important role in steroidogenesis. Dimers containing deglycosylated beta subunit and an alpha subunit lacking either the Asn-52 oligosaccharide or both oligosaccharides fail to stimulate cAMP or steroid formation. Moreover, these derivatives bind to receptor and behave as competitive antagonists. The use of site-directed mutagenesis was critical in uncovering site-specific functions of the hCG N-linked oligosaccharides in signal transduction and reveals the importance of the Asn-52 oligosaccharide in this process.
Matzuk
M M
MM
Department of Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110.
Keene
J L
JL
Boime
I
I
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000031:analogs & derivatives / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D000242:Cyclic AMP / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009844:Oligosaccharides / Q000502:physiology*; D010957:Plasmids; D011374:Progesterone / Q000378:metabolism; D011967:Receptors, Gonadotropin / Q000378:metabolism*; D011974:Receptors, LH / Q000378:metabolism*; D015398:Signal Transduction; D014162:Transfection
D006063:Chorionic Gonadotropin; D009844:Oligosaccharides; D011967:Receptors, Gonadotropin; D011974:Receptors, LH; D011374:Progesterone; D000242:Cyclic AMP; D000262:Adenylyl Cyclases