A SINGLE AMINO-ACID RESIDUE REPLACEMENT IN THE BETA-SUBUNIT OF HUMAN CHORIONIC-GONADOTROPIN RESULTS IN THE LOSS OF BIOLOGICAL-ACTIVITY

The heterodimer, human chorionic gonadotrophin (hCG), contains an alpha-subunit that is common to the glycoprotein hormones and a hormone-specific beta-subunit. A comparison of all known beta-amino acid sequences shows that an aspartic acid at position 99 (with the numbering scheme for hCG-beta) is one of the seven non-Cys invariant residues. Using site-directed mutagenssis we have replaced hCG-beta Asp99 with Arg. Chinese hamster ovary cells, containing a stably integrated gene for bovine alpha-subunit, were transiently transfected with plasmids containing wild-type and mutant hCG-beta cDNAs. The Arg99 beta-mutant associated with the alpha-subunit, but the resulting heterodimer failed to enhance intracellular cyclic AMP production in a gonadotrophin-responsive transformed murine Leydig cell line. Thus, a single amino acid residue replacement in this glycosylated heterodimer containing 237 amino acid residues is sufficient to abolish activity.