On the role of the invariant glutamine at position 54 in the human choriogonadotropin beta subunit.
The twelve Cys and eight of the non-Cys residues are invariant in the glycoprotein hormone beta subunits from a variety of mammalian species. beta-Gin-54 of human lutropin (hLH) and choriogonadotropin (hCG) is one of these invariant amino acid residues. A single A-->G mutation in the LH beta gene of a patient presenting with hypogonadism resulted in the replacement of Gin-54 with Arg [1]. The authors also reported that an expressed mutant of hLH beta, with Arg replacing Gin-54, associated with the alpha subunit, but there was no demonstrable binding of the mutant hormone to receptor. We have replaced Gin-54 in hCG beta with Glu and with Lys using site-directed mutagenesis. The expression plasmids pRSV-hCG beta (wild-type and mutants) were transiently transfected into CHO cells containing a stably integrated gene for bovine alpha, and the media were analyzed for holoproteins, which were characterized in vitro using competitive binding and steroidogenic assays with MA-10 cells. hCG beta(Glu-54) bound to alpha almost as well as hCG beta wild-type, and the resulting heterodimer competed with [125l]hCG binding to the LH/CG receptor and stimulated progesterone production to the same extent as the wild-type control. However, the apparent potencies, as judged by ED50s, were less than those of the wild-type control, the effect being more pronounced in binding than in steroidogenesis. In contrast, hCG beta(Lys-54) associated very poorly with alpha. Our results suggest that while Gin-54 in hCG beta participates in receptor binding, its major function appears to involve alpha binding. Such dual functionality leads to interesting models for holoprotein formation and receptor binding.
Huang
J
J
Department of Biochemistry, University of Georgia, Athens 30602.
Puett
D
D
D000818:Animals; D016466:CHO Cells; D006063:Chorionic Gonadotropin / Q000737:chemistry*; D018997:Chorionic Gonadotropin, beta Subunit, Human; D006224:Cricetinae; D014644:Genetic Variation; D005973:Glutamine / Q000235:genetics*; D008239:Lysine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry*; D013329:Structure-Activity Relationship
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D010446:Peptide Fragments; D005973:Glutamine; D008239:Lysine