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 2ND 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 rec eptors. A previous study on the alpha2-adrenergic receptor demonstrate d that substitution of this acidic residue for the corresponding amide slightly decreases the affinity of the receptor for agonists and comp letely abolishes the effect of Na+ on the affinity for agonists. Since we have previously shown that Na+ modulates the binding affinity of t he LH/CG receptor for ovine LH (oLH) [but not for human CG (hCG)], the experiments described here were designed to determine if the correspo nding residue (D383) of the rat LH/CG receptor also mediates this Naeffect. We used site-directed mutagenesis to create an LH/CG receptor mutant in which D383 was substituted by N. The wild type and mutant re ceptor [designated rLHR(D383N)] were expressed in human embryonic kidn ey 293 cells, and the transfected cells were tested for their ability to bind hCG and oLH in medium containing Na+ or an isoosmolar concentr ation 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 o f the presence or absence of Na+, cells expressing rLHR(D383N) bind oL H with a low affinity comparable to that of the wild type receptor ass ayed in the presence of Na+. We also measured the ability of hCG and o LH to increase cAMP accumulation in cells expressing the wild type and mutant receptors. Since the EC50s for both hormones are one order of magnitude higher in cells expressing rLHR(D383N) than in cells express ing the wild type receptor, our data show that D383 is also important for receptor activation.