title: Effect of modification of the beta-hairpin and long loops simultaneously in both alpha- and beta-subunits on the function of human choriogonadotropin: part II.
abstract: According to the X-ray diffraction, human choriogonadotropin has four beta-hairpin and two long loops, equally distributed in each of the alpha and beta subunits. Radical mutations such as the replacement of alpha 18Phe and alpha 74Phe with Thr in the alpha1 and alpha3 loops respectively and the replacement of alpha 45Lys with Asp in the alpha2 loop in the alpha-subunit were introduced while the loop sequences in the beta-subunit were replaced with the corresponding sequences in hFSH beta. Nine different double mutants with simultaneous mutations in both the alpha and beta loops including hCG alpha1 beta1, hCG alpha1 beta2, hCG alpha1 beta3, hCG alpha2 beta1, hCG alpha2 beta2, hCG alpha2 beta3, hCG alpha3 beta1, hCG alpha3 beta2 and hCG alpha3 beta3 were partially purified from insect High-Five cells. As previously reported (Shao et al., 1996, Mol. Cell. Endocrinol. 122, 173-182), the mutation in the alpha1 loop in the mutant, hCG alpha1 beta, the mutants hCG alpha1 beta1 and hCG alpha1 beta3 caused 200%, increase in the receptor binding, cAMP and progesterone stimulation. The mutant, hCG alpha1 beta2 and all other mutants behaved like the recombinant hCG (rehCG) in the receptor binding and post-receptor signaling activities. The molecular cause for this increase is probably due to a conformational change in the heterodimers caused by the mutation in the alpha1 loop. This conclusion is based on the results of the dissociation studies of the mutants heterodimers which indicated a decreased affinity between the subunits. The first order rate constants for the dissociation of the mutants hCG alpha1 beta1, hCG alpha1 beta2 and hCG alpha beta3 were 3.7 x 10(-2) min(-1), 1.4 x 10(-2) min(-1) and 4.6 x 10(-2) min(-1) respectively, as compared with 4.6 x 10(-3) min(-1) for the rehCG. It seems from the data that alpha 18Phe is located in, or in proximity to the receptor binding site and probably plays a critical role in maintaining either directly or indirectly its conformational integrity.
authors.affiliation: State University of New York at Buffalo, Department of Biological Sciences, 14260, USA.
authors.forename: K
authors.initials: K
authors.lastname: Shao
authors.forename: O P
authors.initials: OP
authors.lastname: Bahl
mesh_terms: D000818:Animals; D002460:Cell Line; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis; D019281:Dimerization; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D007700:Kinetics; D007985:Leydig Cells; D008297:Male; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010053:Ovary / Q000737:chemistry; D011374:Progesterone / Q000096:biosynthesis; D011487:Protein Conformation; D017510:Protein Folding; D011869:Radioligand Assay; D051381:Rats; D011974:Receptors, LH / Q000378:metabolism; D018411:Spodoptera
chemical_list: D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D011374:Progesterone; D000242:Cyclic AMP