Identification of two amino acid residues on the extracellular domain of the lutropin/choriogonadotropin receptor important in signaling.
The lutropin/choriogonadotropin receptor (LH/CG-R) is a G protein-coupled receptor with a relatively large extracellular domain. The cDNAs of LH/CG-R wild type and 15 point and double mutants, which encoded residues of opposite charge to that of wild type, were transiently transfected into COS-7 cells. Human choriogonadotropin (hCG) binding was determined, as was hCG-mediated cAMP production. Most of the replacements resulted in no substantive effect on the binding affinity of hCG to LH/CG-R or on hCG-stimulated cAMP production, although the mutants expressed at a lower level than LH/CG-R wild type. The most interesting observation was noted with two point mutants of LH/CG-R, Glu332-->Lys and Asp333-->Lys, which bound hCG but failed to give increased cAMP production. Several of the mutant forms of LH/CG-R that expressed at low levels were further analyzed by soluble binding assays and Western blots. There was no evidence of any significant degree of intracellular trapping of hCG-binding mutant receptors. The expected major (93 kDa) and minor (78 kDa) forms were found for LH/CG-R wild type and several of the mutants. The Lys235-->Asp and Asp333-->Lys mutants exhibited primarily the lower M(r) form, indicating that receptor processing was impaired or that the mutant higher M(r) form was more rapidly degraded than LH/CG-R wild type. These results demonstrate that Glu332 and Asp333, which are located near the first transmembrane helix, are important in receptor activation, while other conserved ionizable residues of LH/CG-R appear important in cell surface expression or stability but not in binding or signaling.
Huang
J
J
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
Puett
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D006063:Chorionic Gonadotropin / Q000378:metabolism / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism*; D018076:DNA, Complementary; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D051381:Rats; D011974:Receptors, LH / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection
D006063:Chorionic Gonadotropin; D018076:DNA, Complementary; D011974:Receptors, LH; D011994:Recombinant Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; D000242:Cyclic AMP; D008239:Lysine