Lysine residues 2 and 104 of the human chorionic gonadotrophin beta subunit influence receptor binding.
Human chorionic gonadotrophin (hCG), like other members of the glycoprotein hormone family, contains a common alpha subunit and a hormone-specific beta subunit. The latter is a 145 amino acid residue polypeptide with six sites of glycosylation. Positions 2 and 104 are occupied by basic amino acid residues in the 12 known amino acid sequences of mammalian beta subunits from CG and LH, a related gonadotrophin that acts through the same receptor. Lysine residues are found in both these positions in hCG-beta. Using site-directed mutagenesis, each of these two lysines in hCG-beta was replaced with glutamic acid. The mutant and wild-type cDNAs were subcloned into a eukaryotic expression vector, which was then transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for the bovine alpha subunit. Holoprotein formation occurred with each of the two heterologous gonadotrophin mutants, i.e. the bovine alpha subunit bound to hCG-beta (Glu2) and to hCG-beta (Glu104), as well as with the control, i.e. the bovine alpha subunit bound to the hCG-beta wild-type subunit. In two in-vitro assays, one a competitive binding assay with 125I-labelled hCG as bound ligand and the other based on stimulation of progesterone production in a transformed murine Leydig cell line, MA-10, both the heterodimers containing a mutant beta subunit exhibited bioactivity, but their potencies were lower than that of the bovine alpha subunit bound to the hCG-beta wild-type subunit. These results suggest that the basic amino acid residues at positions 2 and 104 in hCG-beta participate, either directly or indirectly, in receptor binding.
Xia
H
H
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Florida 33101.
Huang
J
J
Chen
T M
TM
Puett
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D006063:Chorionic Gonadotropin / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011974:Receptors, LH / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D011994:Recombinant Proteins; D004247:DNA; D008239:Lysine