Effect of modification of all loop regions in the α- and β-subunits of human choriogonadotropin on its signal transduction activity

Abstract

Human choriogonadotropin (hCG), according to its three dimensional structure as determined by X-ray diffraction, has three β-hairpin loops each in the α and β subunit designated as α 1, α 2 α 3 and β 1 β 2 and β 3, respectively. Since similar β-hairpin loops in NGF and TNFβ have been implicated in their direct interaction with the receptor, it prompted the present investigation to determine the role of such loops in receptor binding and post-receptor signaling events in hCG. Based on the three dimensional structure of hCG, radical mutations were introduced in the α loops by replacing hydrophobic α18Phe and α74Phe by hydrophilic Thr residues in the α 1 and α 3 loops, respectively, and positively charged α45Lys by negatively charged Asp in the helical segment in the α 2 loop. The β loops were mutated by replacement of the β 1, β 2 and β 3 sequences with the corresponding hFSH sequences. These replacements included β22Gly, β24Pro and β25Val with Glu, Arg and Phe in β 1, 45Leu Gln Gly Val Leu Pro Ala Leu Pro 53 with Tyr Lys Asn Pro Ala Arg Pro Leu Ile in β 2 and 73Pro Arg Gly with Ala His His in the β 3 loop. Six mutants, hCGα 1β, hCGα 2β and hCGα 3β and hCGαβ 1, hCGαβ 2 and hCGαβ 3, were obtained by co-infection of the insect High-Five cells with baculovirus containing mutant α or β cDNAs and that containing complimentary wild type β or α cDNAs. The mutants were almost completely secreted in the culture medium and were over expressed at levels ranging between 4.5 to 29 μg/ml indicating that mutations had no effect on the secretion or subunit assembly of hCG. In order to remove any contaminating β-subunit, the culture medium was passed through a column of a hCGβ-specific monoclonal antibody, B158. The receptor binding activity of the mutant hCGα 1β, in which α18Phe was replaced with Thr, increased almost 200% relative to rehCG. Similarly, increase in the cAMP and progesterone stimulation by the mutant ranged between 150 to 200%. This increase is believed to be due to a short range conformational change in the mutant as a result of the mutation rather than direct involvement of α18Phe in the receptor binding. The evidence in support of this was derived from the fact that the affinity or interaction between the two subunits was impaired as indicated by the first order rate constant of hCGα 1β ( k m = 4.1 × 10 −2 min −1) at pH 3.0 at 23°C which is one order of magnitude greater relative to rehCG ( k w = 4.6 × 10 −3 min −1). All other mutations had no effect on the receptor binding or signal transduction of hCG indicating that, unlike NGF or TNFβ, β-hairpin loops in hCG were not directly involved in rec binding or post-receptor signaling events. However, since the mutation in the α 1 loop affects the receptor binding site, its presence in the vicinity of the α 1 loop is highly likely.