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.