Effect of modification of the β-hairpin and long loops simultaneously in both α- and β-subunits on the function of human choriogonadotropin: part II

Abstract

According to the X-ray diffraction, human choriogonadotropin has four β-hairpin and two long loops, equally distributed in each of the α and β subunits. Radical mutations such as the replacement of α18Phe and α74Phe with Thr in the α 1 and α 3 loops respectively and the replacement of α45Lys with Asp in the α 2 loop in the α-subunit were introduced while the loop sequences in the β-subunit were replaced with the corresponding sequences in hFSH β. Nine different double mutants with simultaneous mutations in both the α and β loops including hCG α 1 β 1, hCG α 1 β 2, hCG α 1 β 3, hCG α 2 β 1, hCG α 2 β 2, hCG α 2 β 3, hCG α 3 β 1, hCG α 3 β 2 and hCG α 3 β 3 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 α 1 loop in the mutant, hCG α 1 β, the mutants hCG α 1 β 1 and hCG α 1 β 3 caused 200% increase in the receptor binding, cAMP and progesterone stimulation. The mutant, hCG α 1 β 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 α 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 α 1 β 1, hCG α1 β 2 and hCG α 1 β 3 were 3.7×10 −2 min −1, 1.4×10 −2 min −1 and 4.6×10 −2 min −1 respectively, as compared with 4.6×10 −3 min −1 for the rehCG. It seems from the data that α18Phe 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.