MUTATIONS OF ARG(68) OF THE HUMAN CHORIONIC-GONADOTROPIN BETA-SUBUNITLEAD TO REDUCED SECRETION

The Arg(68)-Leu(69) sequence is invariant in the beta subunits of chor ionic gonadotrophin and luteinizing hormone from a variety of species. Using site-directed mutagenesis of the human chorionic gonadotrophin (hCG)-beta cDNA, several replacements of Arg(68), an Ala replacement o f Leu(69), and a multiple replacement with Ala-Ala-Ala-Ala of the tetr apeptide sequence, Arg(68)Leu(69)-Pro(70)-Gly(71), were prepared and c haracterized. The wild-type and mutant cDNAs were subcloned into a pRS V expression vector and transiently transfected into CHO cells contain ing a stably integrated gene for bovine alpha. Concentrations of secre ted wild-type and mutant hCG-beta subunit and holoprotein were determi ned using radioimmunoassays; potencies, i.e. the ratio of biologic to immunologic activity, of several of the mutant heterodimers were measu red in vitro via gonadotrophin-mediated steroidogenesis in transformed murine Leydig cells (MA-10). The Leu(69)-->Ala mutant formed a mutant holoprotein that was essentially equipotent with wild-type hormone in the steroidogenesis assay. The Arg(68) replacements with Lys, Ala, an d Leu were poorly secreted by the cells, e.g. <10% that of wild-type h CG; however, sufficient quantities of mutant holoproteins containing L ys(68) and Ala(68) were obtained for biological assays, and both exhib ited greater apparent potencies than wildtype hormone. Likewise, a mut ant holoprotein containing the Arg(68)-Leu(69)-Pro(70)-Gly(71)-->Ala-A la-Ala-Ala multiple replacement was apparently more potent than wild-t ype hormone, but it too was secreted at lower levels than wild-type. T hese results establish that replacements of Arg(68) in hCG-beta dimini sh secretion, but the small amount of holoprotein that is formed and s ecreted appears to be of somewhat greater potency than wild-type hormo ne.