Replacement of the invariant tyrosine in the CAGY region of the human chorionic gonadotropin beta subunit.
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 hCG beta, 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 hCG beta(Phe-37) mutant bound to alpha essentially to the same extent as hCG beta wild-type, while the hCG beta(Leu-37) mutant formed somewhat less heterodimer. The heterologous heterodimeric mutant and wild-type gonadotropins were equipotent in a competitive binding assay with [125I]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 hCG beta without any dramatic effect on function. These results indicate that Tyr-37 in hCG beta, 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.
Xia
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Reproductive Sciences and Endocrinology Laboratory, University of Miami School of Medicine, FL 33101.
Fernandez
L M
LM
Puett
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D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002461:Cell Line, Transformed; D006063:Chorionic Gonadotropin / Q000235:genetics*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D006224:Cricetinae; D006801:Humans; D007985:Leydig Cells / Q000187:drug effects; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014443:Tyrosine