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A Functional Determinant in Human Luteinizing Hormone and Chorionic Gonadotropin: Differential Effect of Mutations about ß-GLN-54¹

Endocrine Unit and Reproductive Endocrine Sciences Center (S.-B.H., L.J., H.T.K.), Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and the Department of Laboratory Medicine and Pathology (P.C.R.) Mayo Medical School, Rochester, Minnesota 55905

Address all correspondence and requests for reprints to: Henry T. Keutmann, M.D., Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: Keutmann{at}helix.mgh.harvard.edu

There is evidence that the conserved glutamine at residue 54 in the ß-subunit of human LH and and CG (hCG) is important for biological activity. Mutation to Arg in LH has been reported to impair receptor binding, leading to a documented case of hypogonadism, whereas in hCG the mutation has been shown to result in defective subunit association. Functional distinctions between LH and hCG have been described, but the significance of peptide-chain differences between the two has not been investigated systematically. We therefore compared the role of Gln-54 and its neighboring residues in both hormones, through replacement by amino acids with contrasting properties using site-directed mutagenesis. The mutant subunits were coexpressed with -subunit in mammalian (Chinese hamster ovary) cells and the secreted hormones assayed for heterodimer formation, receptor binding, and steroidogenesis in murine Leydig cell tumor (MA-10) cells. Basic (Arg, Lys) substitution for Gln-54 in either hormone markedly impaired subunit association (<20% of wild-type) and the heterodimers that were formed were inactive (<5% of wild-type) in both assays. Arg-substituted hCG was also inactive in an adenylate cyclase assay using HEK-293 cells expressing rat LH/hCG receptor. After acidic (Glu) or neutral (Ala) substitution, heterodimer formation was less impaired (50–60% of wild-type), but effects on receptor interaction differed between the two hormones. The LH mutants still lacked binding activity, whereas the hCG products were fully active. The importance of residue 54 for receptor interaction appears to be sharply localized because mutation at adjacent positions (Pro-53 and Val-55) did not impair the activity of either hormone. Diminished heterodimer formation by Ile-53 mutation in LH (but not hCG), together with the similar effects of basic mutations at 54, imply long-distance effects as these residues are remote from in the crystal structure. Our findings indicate that position 54 in LH and hCG is a determinant for both subunit association and receptor interaction. The differing responses between LH and hCG to certain mutations suggest that structural characteristics of the peptide chains may confer functional differences despite their close sequence homology.

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