REPLACEMENT OF THE INVARIANT TYROSINE IN THE CAGY REGION OF THE HUMAN CHORIONIC-GONADOTROPIN BETA SUBUNIT

[摘要] The mammalian glycoprotein hormone beta subunits contain a highly conserved amino acid sequence, Cys-Ala-Gly-Tyr-Cys (residues 34-38 of human chorionic gonadotropin beta), that is denoted as the 'CAGY region'. Using site-directed mutagenesis we have replaced Tyr-37 in hCGbeta, i.e., the invariant Tyr in all known mammalian CG, LH, FSH, and TSH beta subunits, with two hydrophobic amino acid residues, Phe and Leu. The resultant mutant forms were characterized for alpha subunit binding and the resulting heterodimers were analyzed for biological activity using two in vitro assays with transformed murine Leydig cells (MA-10). Chinese hamster ovary cells containing a stably integrated gene for bovine alpha were transiently transfected with a eukaryotic expression vector containing a Rous sarcoma viral promoter and the wild-type and mutant cDNAs. The hCGbeta(Phe-37) mutant bound to alpha essentially to the same extent as hCGbeta wild-type, while the hCGbeta(Leu-37) mutant formed somewhat less heterodimer. The heterologous heterodimeric mutant and wild-type gonadotropins were equipotent in a competitive binding assay with [I-125]hCG. In a steroidogenic assay, the mutant hormones were active, but they appeared slightly less potent than the wild-type form. Thus, this invariant Tyr can be replaced with another aromatic amino acid residue or with a hydrophobic, but not aromatic amino acid residue in hCGbeta without any dramatic effect on function. These results indicate that Tyr-37 in hCGbeta, while not obligatory, may participate, either directly or indirectly, in subunit assembly and that the hydroxyl group may function in a modulatory role in signaling.

[发布日期] 1993-03-01

[关键词] GONADOTROPIN;BETA-SUBUNIT;MUTAGENESIS;STEROIDOGENESIS