CONTRIBUTIONS OF ARGININE-43 AND ARGININE-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 a 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 I-125-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.