The regulation of the binding affinity of the luteinizing hormone/choriogonadotropin receptor by sodium ions is mediated by a highly conserved aspartate located in the second transmembrane domain of G protein-coupled receptors.
Sequence alignment shows that there is a highly conserved aspartate in the second transmembrane helix of virtually all G protein-coupled receptors. A previous study on the alpha 2-adrenergic receptor demonstrated that substitution of this acidic residue for the corresponding amide slightly decreases the affinity of the receptor for agonists and completely abolishes the effect of Na+ on the affinity for agonists. Since we have previously shown that Na+ modulates the binding affinity of the LH/CG receptor for ovine LH (oLH) [but not for human CG (hCG)], the experiments described here were designed to determine if the corresponding residue (D383) of the rat LH/CG receptor also mediates this Na+ effect. We used site-directed mutagenesis to create an LH/CG receptor mutant in which D383 was substituted by N. The wild type and mutant receptor [designated rLHR(D383N)] were expressed in human embryonic kidney 293 cells, and the transfected cells were tested for their ability to bind hCG and oLH in medium containing Na+ or an isoosmolar concentration of an appropriate sodium substitute. The results presented here show that this single point mutation of the LH/CG receptor leads to a slight reduction in affinity for hCG and oLH but completely abolishes the effects of Na+ removal on the affinity for oLH. Thus, regardless of the presence or absence of Na+, cells expressing rLHR(D383N) bind oLH with a low affinity comparable to that of the wild type receptor assayed in the presence of Na+. We also measured the ability of hCG and oLH to increase cAMP accumulation in cells expressing the wild type and mutant receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
Quintana
J
J
Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242.
Wang
H
H
Ascoli
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D017124:Conserved Sequence; D000242:Cyclic AMP / Q000378:metabolism; D004622:Embryo, Mammalian; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney; D007986:Luteinizing Hormone / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D011974:Receptors, LH / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology*; D014162:Transfection
D006063:Chorionic Gonadotropin; D011974:Receptors, LH; D001224:Aspartic Acid; D007986:Luteinizing Hormone; D012964:Sodium; D000242:Cyclic AMP; D019204:GTP-Binding Proteins