Role of the invariant aspartic acid 99 of human choriogonadotropin beta in receptor binding and biological activity.
The four human glycoprotein hormones are heterodimers that contain a common alpha subunit and a hormone-specific beta subunit. Within this hormone family, 23 amino acid sequences from 11 mammalian species are available. There are 19 invariant amino acid residues in the beta subunits, 12 of which are Cys that form six disulfide bonds. Of the remaining seven conserved amino acid residues, we have investigated the role of an Asp which occurs at position 99 in human choriogonadotropin beta (hCG beta). Site-directed mutagenesis was used to replace hCG beta Asp99 with three residues, Glu, Asn, and Arg, and to prepare an inversion double mutant protein, Arg94----Asp and Asp99----Arg. The cDNAs were placed in a eukaryotic expression vector, and the plasmids were transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Radioimmunoassays demonstrated that the mutant forms of hCG beta were capable of subunit assembly to the same extent as hCG beta wild type. The heterologous heterodimers were assayed in vitro using transformed mouse Leydig cells (MA-10) by competitive inhibition of 125I-hCG binding and stimulation of progesterone production. The gonadotropins containing Glu and Asn were active, although the potency was less than that associated with the hCG beta wild type-containing gonadotropin. In contrast, the Arg99-containing mutant protein and the inversion mutant protein Asp94/Arg99 were devoid of activity. Thus, in hCG beta Asp99 can be substituted with certain residues without total loss of function, although replacement with a positively charged residue leads to an inactive heterodimer. The primary role of Asp99 in hCG beta seems to involve, either directly or indirectly, receptor recognition.
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
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Chen
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Wang
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Puett
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics*; D001483:Base Sequence; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000235:genetics* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011863:Radioimmunoassay; D014162:Transfection
D006063:Chorionic Gonadotropin; D001224:Aspartic Acid; D004247:DNA; D001120:Arginine