Identification of ionizable amino acid residues on the extracellular domain of the lutropin receptor involved in ligand binding

The LH receptor (LHR) is a G protein-coupled receptor characterized by a re latively large N-terminal extracellular domain responsible for high affinit y ligand binding. Based on a model proposed for a major portion of the extr acellular domain that contains a number of leucine-rich repeats, nine ioniz able amino acid residues (Glu(57), Glu(80), Lys(158) Glu(181), Lys(183), Gl u(184), Glu(188), Lys(190), and Asp(206)) were selected for charge reversal mutagenesis based on their locations in the proposed model and their poten tial to serve as ligand contact sites. Mutant LHR complementary DNAs were t ransiently transfected into COS-7 cells, and the expressed receptors were c haracterized by Western blot analysis, competitive ligand (hCG) binding, an d ligand-mediated cAMP production. The most interesting mutants were K158E, K183E, E184K and D206K, which were present on the plasma membrane fraction , as judged by Western blots, but were incapable of binding hCG and, of cou rse, were deficient in hCG-mediated cAMP production. Other replacements at these positions, K158R,Q,G; K183R, Q,G; E184N; and D206E,Q, led to cell sur face binding and signaling. The mutants E57K, E189K and K190E behaved simil arly to wild-type LHR; E80K was trapped intracellularly, but bound ligand i n solubilized cells; and E181K was not expressed or was rapidly degraded. T hese results, based on 18 point mutants of LHR, indicate that Lys(158) Lys( 183), Glu(184), and Asp(206) are involved, either directly or indirectly, i n gonadotropin binding and support the general nature of the proposed model .