THE CARBOXY-TERMINAL REGION OF THE GLYCOPROTEIN HORMONE ALPHA-SUBUNIT - CONTRIBUTIONS TO RECEPTOR-BINDING AND SIGNALING IN HUMAN CHORIONIC-GONADOTROPIN

The glycoprotein hormones are heterodimeric and contain a common alpha-subunit, which is noncovalently associated with a hormone-specific beta-subunit. The alpha-subunit has been highly conserved throughout evolution; for example, the five amino acid residues of the carboxy-terminus, Tyr-Tyr-His-Lys-Ser-COOH, are identical in nine of the 10 available amino acid sequences. it has been shown that enzymatic removal of these five amino acid residues, while not affecting holoprotein formation, results in a heterodimer that exhibits very little, if any, binding to the CG/LH receptor. Using site-directed mutagenesis on the human alpha-subunit, we have prepared two deletion mutants, Des-(88-92)alpha and Des-(89-92)alpha, and two point mutants, where each of the two tyrosines, 88 and 89, was replaced with phenylalanine, in order to delineate more specifically the contributions of these aromatic side-chains to receptor binding. The cDNAs for wild-type hCG-alpha and mutants were introduced into a pcDNAINEO expression vector, and the cDNA for hCG-beta was inserted into a pRSV plasmid; both were transiently cotransfected into DUXB-11 cells. The media were collected, and RIAs showed that all mutants formed heterodimers; moreover, there was no discernable difference in subunit assembly between wild-type hCG-alpha and the various mutant alpha-subunits. The gonadotropin mutants were assayed in vitro using a competitive binding assay with [I-125]hCG and stimulation of progesterone production in the transformed murine Leydig cell line MA-10. Consistent with the observations of others, [Des-(88-92)alpha-beta-wild-type] hCG was devoid of activity; in contrast, [Des-(89-92)alpha-beta-wild-type]hCG retained 15-30% of the activity of expressed wild-type hCG. Phe replacements of Tyr-88 and Tyr-89 yielded heterodimers with reduced binding affinity for the receptor relative to wild-type hCG; that associated with [(Phe89)alpha-beta-wild-type] hCG was the lowest. Interestingly, at saturating concentrations, [(Phe88)alpha-beta-wild-type]hCG produced a significantly higher level of progesterone than did wild-type hCG. These results imply important roles for both tyrosines 88 and 89 of the alpha-subunit of hCG in receptor binding and activation. The hydroxyl group on Tyr89 seems to contribute more to receptor binding than activation, while that on Tyr88 appears to attenuate the maximal steroidogenic response. These results indicate that hCG-alpha-(1-88) is the shortest amino-terminal fragment, when combined with hCG-beta capable of receptor binding and activation.