Effect of modification of all loop regions in the alpha- and beta-subunits of human choriogonadotropin on its signal transduction activity.
Human choriogonadotropin (hCG), according to its three dimensional structure as determined by X-ray diffraction, has three beta-hairpin loops each in the alpha and beta subunit designated as alpha 1, alpha 2 alpha 3 and beta 1 beta 2 and beta 3, respectively. Since similar beta-hairpin loops in NGF and TNF beta 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 alpha loops by replacing hydrophobic alpha 18Phe and alpha 74Phe by hydrophilic Thr residues in the alpha 1 and alpha 3 loops, respectively, and positively charged alpha 45Lys by negatively charged Asp in the helical segment in the alpha 2 loop. The beta loops were mutated by replacement of the beta 1, beta 2 and beta 3 sequences with the corresponding hFSH sequences. These replacements included beta 22Gly, beta 24Pro and beta 25Val with Glu, Arg and Phe in beta 1, 45Leu Gln Gly Val Leu Pro Ala Leu Pro53 with Tyr Lys Asn Pro Ala Arg Pro Leu Ile in beta 2 and 73Pro Arg Gly with Ala His His in the beta 3 loop. Six mutants, hCG alpha 1 beta, hCG alpha 2 beta and hCG alpha 3 beta and hCG alpha beta 1, hCG alpha beta 2 and h CG alpha beta 3, were obtained by co-infection of the insect High-Five cells with baculovirus containing mutant alpha or beta cDNAs and that containing complimentary wild type beta or alpha cDNAs. The mutants were almost completely secreted in the culture medium and were over expressed at levels ranging between 4.5 to 29 micrograms/ml indicating that mutations had no effect on the secretion or subunit assembly of hCG. In order to remove any contaminating beta-subunit, the culture medium was passed through a column of an hCG beta-specific monoclonal antibody, B158. The receptor binding activity of the mutant hCG alpha 1 beta, in which alpha 18Phe 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 alpha 18Phe 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 hCG alpha 1 beta (km = 4.1 x 10(-2) min-1) at pH 3.0 at 23 degrees C which is one order of magnitude greater relative to rehCG (kw = 4.6 x 10(-3) min-1). All other mutations had no effect on the receptor binding or signal transduction of hCG indicating that, unlike NGF or TNF beta, beta-hairpin loops in hCG were not directly involved in receptor binding or post-receptor signaling events. However, since the mutation in the alpha 1 loop affects the receptor binding site, its presence in the vicinity of the alpha 1 loop is highly likely.
State University of New York at Buffalo, Department of Biological Sciences 14260, USA.
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D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D006063:Chorionic Gonadotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011974:Receptors, LH / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014162:Transfection
D006063:Chorionic Gonadotropin; D018997:Chorionic Gonadotropin, beta Subunit, Human; D018076:DNA, Complementary; D015292:Glycoprotein Hormones, alpha Subunit; D046911:Macromolecular Substances; D011974:Receptors, LH