Contributions of arginines-43 and -94 of human choriogonadotropin beta to receptor binding and activation as determined by oligonucleotide-based mutagenesis.
Members of the glycoprotein hormone family contain a common alpha subunit and a hormone-specific beta subunit. Human choriogonadotropin (hCG) beta is a 145 amino acid residue protein glycosylated at 6 positions (2 N-linked and 4 O-linked oligosaccharides). In an effort to elucidate receptor determinants on hCG beta, we have used site-directed mutagenesis to prepare and express several mutant cDNAs with replacements at arginines-43 and -94. Arg-43 is invariant in all known mammalian CG/lutropin beta amino acid sequences, and Arg-94 is conserved in 10 of the 12 sequences. Moreover, various studies involving synthetic peptides and enzymatic digestions of intact beta chains suggest that these residues may be important in hCG receptor binding. Point mutants were made in which these two arginines were replaced with the corresponding residues in human follitropin beta, Leu-43 and Asp-94. The wild-type and mutant beta chains were expressed in CHO cells containing a stably integrated gene for bovine alpha, and heterodimer formation occurred. These heterologous gonadotropins were active in assays using transformed Leydig cells, competitive binding with standard 125I-hCG, and cAMP and progesterone production, but the potency was considerably less than that associated with the hCG beta wild-type-containing gonadotropin. The double-mutant protein Arg-43 to Leu/Arg-94 to Asp also associated with bovine alpha, but the resultant heterodimer exhibited only low activity. Replacement of each arginine with lysine yielded heterodimers that were at least as potent as bovine alpha-hCG beta wild type, but the Lys-43-containing beta chain appeared to exhibit a low degree of subunit association or reduced stability relative to the expressed hCG beta wild type. These results demonstrate that arginines-43 and -94 contribute to receptor binding through a positive charge.
Chen
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Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Puett
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D
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics*; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D007984:Leydig Cell Tumor / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010053:Ovary; D011974:Receptors, LH / Q000737:chemistry / Q000235:genetics*; D014407:Tumor Cells, Cultured
D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D001120:Arginine