The carboxy-terminal region of the glycoprotein hormone α-subunit: Contributions to receptor binding and signaling in human chorionic gonadotropin

Abstract

The glycoprotein hormones are heterodimeric and contain a common α-subunit, which is noncovalently associated with a hormone-specific β-subunit. The α-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 α-subunit, we have prepared two deletion mutants, Des-(88-92)α and Des-(89-92)α, 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 hCGα and mutants were introduced into a pcDNAINEO expression vector, and the cDNA for hCGβ 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 hCGα and the various mutant α-subunits. The gonadotropin mutants were assayed in vitro using a competitive binding assay with [125I]hCG and stimulation of progesterone production in the transformed murine Leydig cell line MA-10. Consistent with the observations of others, [Des-(88-92)α-β wild-type]hCG was devoid of activity; in contrast, [Des-(89-92)α-β wild-type]hCG retained 15-30% of the activity of expressed wild-type hCG. Phe replacements of Tyr88 and Tyr89 yielded heterodimers with reduced binding affinity for the receptor relative to wild-type hCG; that associated with [(Phe89)α-β wild-type]hCG was the lowest. Interestingly, at saturating concentrations, [(Phe88)α-β 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 α-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 hCGα-(1-88) is the shortest amino-terminal fragment, when combined with hCGβ, capable of receptor binding and activation.