Conversion of lysine 91 to methionine or glutamic acid in human choriogonadotropin alpha results in the loss of cAMP inducibility.
Human choriogonadotropin (hCG) is a heterodimeric glycoprotein hormone. The alpha subunit comprises 92 amino acids, of which 6 are Lys residues (Morgan, F.G., Birken, S., and Canfield, R.E. (1975) J. Biol. Chem. 250, 5247-5258). Our photoaffinity-labeling studies indicate that several of these Lys residues make contact with the lutropin receptor and are covalently cross-linked to the receptor. Lys-91 of the alpha subunit is of interest because deletion of the two alpha C-terminal residues, Lys-91 and Ser-92, results in a significant reduction in the bioactivity of lutropin and thyrotropin (Cheng, K.-W., Glazer, A.N., and Pierce, J.G. (1973) J. Biol. Chem. 248, 7930-7937). To determine the importance of Lys-alpha 91, we substituted it with Arg, Met, or Glu. The resulting mutant alpha cDNA constructs were co-transfected into CHO cells with the wild type hCG beta cDNA construct. Secreted hCG dimers were assayed for binding to receptors on porcine granulosa cells and stimulation of cAMP synthesis in a murine Leydig tumor cell line. The natural hCG, wild type hCG, and all mutant hCGs recognized the receptor, although with somewhat divergent affinities. However, there was a striking difference in the ability of cAMP induction. The natural hCG, wild type hCG, and Lys-91----Arg mutant hCG induced cAMP synthesis, whereas the Lys-91----Met and Lys-91----Glu mutants did not. These results demonstrate that Lys-91 is important for receptor modulation in the stimulation of cAMP synthesis.
Yoo
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Department of Molecular Biology, University of Wyoming, Laramie 82071-3944.
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D000818:Animals; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000096:biosynthesis* / Q000378:metabolism; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005971:Glutamates / Q000378:metabolism*; D018698:Glutamic Acid; D008239:Lysine / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D011863:Radioimmunoassay; D013552:Swine; D014162:Transfection; D014407:Tumor Cells, Cultured
D006063:Chorionic Gonadotropin; D005971:Glutamates; D018698:Glutamic Acid; D004247:DNA; D008715:Methionine; D000242:Cyclic AMP; D008239:Lysine