Delineation via site-directed mutagenesis of the carboxyl-terminal region of human choriogonadotropin beta required for subunit assembly and biological activity.
The choriogonadotropin beta subunit is unique in the human glycoprotein hormone family in containing a carboxyl-terminal extension, with four sites of O-glycosylation, that is not present in the other beta subunits. We have used site-directed mutagenesis to define boundaries on the extent to which truncations can be made at the COOH terminus without abolishing subunit assembly and biological activity. Two COOH-terminal deletion mutant chains of human choriogonadotropin beta, des(93-145) and des(101-145), were prepared and expressed in Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. The heterologous gonadotropin, bovine alpha-human choriogonadotropin des(101-145) beta, formed a heterodimer and, when assayed with transformed murine Leydig cells in vitro, competed with the binding of standard human choriogonadotropin and stimulated both cAMP and progesterone production, albeit with a reduced potency relative to bovine alpha-human choriogonadotropin beta wild type. In contrast, human choriogonadotropin des(93-145) beta, expressed under identical conditions in the presence of bovine alpha, failed to form heterodimer and thus exhibited no competitive binding and was without effect on cAMP and progesterone levels. Consequently, removal of the putative determinant loop region of the beta subunit (residues 93-100), which is believed to be important in determining receptor specificity, abolishes association with alpha. Hence, in addition to its possible role as a receptor determinant, this region of the molecule appears to be critical for proper folding or subunit interaction. The truncated form of human choriogonadotropin beta lacking residues 101-145 is the shortest form of the subunit yet described that retains biological activity. Moreover, these results demonstrate that the proposed disulfide between Cys-26 and Cys-110 is not required for subunit assembly or for receptor binding and subsequent intracellular signaling.
Chen
F
F
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Puett
D
D
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000235:genetics* / Q000494:pharmacology; D018997:Chorionic Gonadotropin, beta Subunit, Human; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D007985:Leydig Cells; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics* / Q000494:pharmacology; D010957:Plasmids; D011374:Progesterone / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D010446:Peptide Fragments; D011994:Recombinant Proteins; D011374:Progesterone; D004247:DNA; D015056:1-Methyl-3-isobutylxanthine