Lys583 in the third extracellular loop of the lutropin/choriogonadotropin receptor is critical for signaling.
The lutropin/choriogonadotropin receptor (LH/CG-R) contains a relatively large extracellular domain, in addition to the seven transmembrane helices (TMH), three extracellular loops (ECL), and three intracellular loops typical of G protein-coupled receptors. While high affinity ligand binding has been attributed to the N-terminal extracellular domain, there is evidence that portions of the three ECLs may function in ligand binding and transmembrane signaling. We have investigated the role of several ionizable amino acid residues of rat LH/CG-R in human choriogonadotropin (hCG) binding and hCG-mediated cAMP production. COS-7 cells were transfected with the pSVL expression vector containing cDNAs of either wild-type or mutant rat LH/CG-R. Several point mutants of Lys583, located at the interface of ECL III and TMH VII, bound hCG like wild-type receptor but exhibited greatly diminished ligand-mediated signaling. Neither the point mutant, Lys401-->Asp (ECL I), nor the double mutant, Asp397-->Lys/Lys583-->Asp (ECLs I and III, respectively, showed significant hCG binding to intact cells; in detergent-solubilized cells, only the double mutant bound hCG. The mutants Arg341-->Glu (interface of the extracellular domain and TMH I) and Lys488-->Glu (ECL II) proved to be similar to wild-type receptor in binding and signaling. Our results establish that Lys583 is important in signaling but not ligand binding. Its location on the opposite side of the membrane from GS precludes a direct interaction, thus emphasizing the importance of a conformational change in the receptor and suggesting that ligand binding to receptor and ligand-mediated receptor activation are dissociable phenomena.
Fernandez
L M
LM
Department of Biochemistry and Molecular Biology, University of Georgia, Athens 30602, USA.
Puett
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D018014:Gene Transfer Techniques; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017354:Point Mutation; D051381:Rats; D011974:Receptors, LH / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D018076:DNA, Complementary; D011974:Receptors, LH; D008239:Lysine