Site-directed mutagenesis of the human chorionic gonadotropin beta-subunit: bioactivity of a heterologous hormone, bovine alpha-human des-(122-145)beta.
Human CG contains an alpha-subunit, common to the pituitary glycoprotein hormones, and a hormone-specific beta-subunit, but unlike the pituitary beta-subunits, hCG beta is characterized by an O-glycosylated carboxy-terminal extension. A mutant beta-subunit, des-(122-145)hCG beta, was prepared using site-directed mutagenesis, and the pRSV expression plasmids were transfected into Chinese hamster ovary cells that produce the bovine alpha-subunit (b alpha). The mutant beta-subunit binds to b alpha, and the heterologous gonadotropin, b alpha-des-(122-145)hCG beta, was capable of stimulating steroidogenesis in cultured Leydig tumor cells (MA-10) to the same extent as standard hCG. When compared with the heterologous gonadotropin, b alpha-hCG beta wild type, the hybrid hormone with the truncated hCG beta exhibited equal potency, within the accuracy of the RIAs used to determine hormone concentrations, and gave a similar time course of steroidogenesis. Interestingly, these transformed Leydig cells do not distinguish between the steroidogenic potencies (as measured by progesterone production) of hCG and human LH (hLH) as do some preparations of normal rodent Leydig cells (as measured by testosterone production). However, the MA-10 cells were able to distinguish hCG from hLH based on their cAMP response; the latter produced a greater response at both maximal and submaximal gonadotropin concentrations. The two expressed heterologous gonadotropins were equipotent in their abilities to stimulate cAMP and gave similar time courses of cAMP accumulation in MA-10 cells. Thus, the carboxy-terminal extension of hCG beta is not required for association with the alpha-subunit nor for functional receptor binding, as judged by cAMP accumulation and progesterone production in MA-10 cells.
el-Deiry
S
S
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Kaetzel
D
D
Kennedy
G
G
Nilson
J
J
Puett
D
D
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000235:genetics* / Q000494:pharmacology; D018997:Chorionic Gonadotropin, beta Subunit, Human; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000096:biosynthesis; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000235:genetics*; D011374:Progesterone / Q000096:biosynthesis; D011863:Radioimmunoassay; D014162:Transfection
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; C062304:HCG-beta, des-(122-145)-; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D011374:Progesterone; D000242:Cyclic AMP