Identification of conserved amino acid residues in the beta subunit of human choriogonadotropin important in holoprotein formation.
The beta subunits of mammalian glycoprotein hormones contain 20 invariant amino acid residues, including 12 Cys residues that form six disulfide bonds, as well as other highly conserved residues. Such stringent conservation suggests an important role for those residues in chain folding, holoprotein formation, or receptor binding/activation, but not receptor specificity. Using site-directed mutagenesis, we have prepared and characterized replacements of 14 conserved non-Cys residues in human choriogonadotropin beta for which there is little, if any, information available: Pro7, Thr32, Tyr37, Thr40, Val56, Tyr59, Phe64, Pro70, Gly71, Ser81, Tyr82, Val84, Ala85, and Ser87. The cDNAs were subcloned into a pRSV mammalian expression vector and transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Holoprotein formation was assessed by radioimmunoassays, and in vitro competitive binding and steroidogenic assays were used to determine potencies. Our results demonstrated that Tyr37, Thr40, Tyr59, and Ala85 participate directly or indirectly in holoprotein formation, as may Ser81. Replacements of the other conserved amino acid residues resulted in no significant change in subunit assembly or receptor binding/activation as measured, although an Arg at position 81 or 82 reduced secretion. This study has identified several amino acid residues of the beta subunit that are important in alpha binding and has raised interesting questions on the constraints that maintain conservation of amino acid residues during evolution.
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Department of Biochemistry, University of Georgia, Athens 30602.
Puett
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D016466:CHO Cells; D002417:Cattle; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006224:Cricetinae; D004247:DNA; D006801:Humans; D007985:Leydig Cells / Q000166:cytology; D008297:Male; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011374:Progesterone / Q000096:biosynthesis; D011863:Radioimmunoassay
D000596:Amino Acids; D006063:Chorionic Gonadotropin; D011374:Progesterone; D004247:DNA