Effect of modification of the beta-hairpin and long loops simultaneously in both alpha- and beta-subunits on the function of human choriogonadotropin .2.

[摘要] 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 alpha(1) and alpha(3) loops respectively and the replacement of alpha 45Lys with Asp in the alpha(2) 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 alpha(1) beta(1), hCG alpha(1) beta(2), hCG alpha(1) beta(3), hCG alpha(2) beta(1), hCG alpha(2) beta(2), hCG alpha(2) beta(3), hCG alpha(3) beta(1), hCG alpha(3) beta(2) and hCG alpha(3) beta(3) were partially purified from insect High-Five cells. As previously reported (Shao et al., 1996, Mel. Cell. Endocrinol. 122, 173-182), the mutation in the alpha(1) loop in the mutant, hCG alpha(1) beta, the mutants hCG alpha(1) beta(1) and hCG alpha(1) beta(3) caused 200% increase in the receptor binding, cAMP and progesterone stimulation. The mutant, hCG alpha(1) beta(2) 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 alpha(1) 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 alpha(1) beta(1), hCG alpha(1) beta(2) and hCG alpha(1) beta(3) 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. (C) 1997 Elsevier Science Ireland Ltd.

[发布日期] 1997-03-28

[关键词] choriogonadotropin;site directed mutagenesis;receptor binding;signal transduction