Identification of amino acid residues in transmembrane helices VI and VII of the lutropin/choriogonadotropin receptor involved in signaling.
The lutropin/chroriogonadotropin receptor (LH/CG-R) is a member of the G protein-coupled receptor superfamily containing a relatively large extracellular domain responsible for high affinity ligand binding. There is a paucity of information on the mechanism of ligand-mediated transmembrane signaling via the seven transmembrane helices (TMH) of these receptors. In the present study we have used site-directed mutagenesis to replace each of nine conserved amino acid residues in the TMHs of rat LH/CG-R. COS-7 cells were transiently transfected with the eukaryotic expression vector pSVL-LH/CG-R, wild-type and mutants, followed by measurements of human CG binding and human CG-mediated cAMP production. Three point mutants of LH/CG-R were prepared that diminished signaling but not binding: Pro-562-Leu (TMH VI), Pro-591-Leu(TMH VII), and Tyr-601-Ala (TMH VII). Two point mutants of LH/CG-R, Pro-479-Leu(TMH IV) and Pro-598-Leu(TMH VII), resulted in impaired localization, and four receptor mutants, Thr-424-Ala (TMH III), Ser-562-Ala (TMH VI), Met-560-Leu (TMH VI), and Tyr-590-Ala (TMH VII), were similar to wild-type LH/CG-R. In summary, these findings indicate a critical role of Tyr-601 in transmembrane signaling of LH/CG-R since an Ala replacement results in almost total abolition of cAMP production in response to human CG; prolines 562 and 591 also appear to be important for full signaling.
Fernandez
L M
LM
Department of Biochemistry & Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Puett
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D006063:Chorionic Gonadotropin / Q000378:metabolism* / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D011974:Receptors, LH / Q000737:chemistry* / Q000302:isolation & purification / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D014162:Transfection
D006063:Chorionic Gonadotropin; D018076:DNA, Complementary; D011974:Receptors, LH; D011994:Recombinant Proteins; D000242:Cyclic AMP; D019204:GTP-Binding Proteins