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 relatively large N-terminal extracellular domain responsible for high affinity ligand binding. Based on a model proposed for a major portion of the extracellular domain that contains a number of leucine-rich repeats, nine ionizable amino acid residues (Glu57, Glu80, Lys158, Glu181, Lys183, Glu184, Glu188, Lys190, and Asp206) were selected for charge reversal mutagenesis based on their locations in the proposed model and their potential to serve as ligand contact sites. Mutant LHR complementary DNAs were transiently transfected into COS-7 cells, and the expressed receptors were characterized by Western blot analysis, competitive ligand (hCG) binding, and 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 course, 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 surface binding and signaling. The mutants E57K, E189K, and K190E behaved similarly to wild-type LHR; E80K was trapped intracellularly, but bound ligand in solubilized cells; and E181K was not expressed or was rapidly degraded. These results, based on 18 point mutants of LHR, indicate that Lys158, Lys183, Glu184, and Asp206 are involved, either directly or indirectly, in gonadotropin binding and support the general nature of the proposed model.
Bhowmick
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N
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602-7229, USA.
Narayan
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P
Puett
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D
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D005110:Extracellular Space / Q000378:metabolism*; D007477:Ions; D008024:Ligands; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation / Q000502:physiology; D011974:Receptors, LH / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D000596:Amino Acids; D007477:Ions; D008024:Ligands; D011974:Receptors, LH; D000242:Cyclic AMP