EVIDENCE FOR AN IMPORTANT FUNCTIONAL-ROLE OF INTRACELLULAR LOOP-II OFTHE LUTROPIN RECEPTOR

The lutropin receptor (LHR) is a G protein-coupled receptor in which h igh affinity ligand binding occurs to the relatively large extracellul ar N-terminal domain. Various portions of the receptor have been mappe d for their relative importance in localization and in hormone-mediate d signaling. There is, however, a paucity of information available on the intracellular loops (ICL), where, along with the C-terminal cytopl asmic tail, G protein coupling is expected to occur. Site-directed mut agenesis was used to investigate the role of several conserved ionizab le groups and one tyrosyl residue in ICLs I-III of the rat LHR. The pS VL expression vector, containing the LHR cDNA (wild-type and mutants), was transiently transfected into COS-7 cells, and human choriogonadot ropin (hCG) binding and hCG-mediated cAMP production were determined. Several point mutants of amino acid residues in ICL II were prepared a nd characterized with the following results: replacements of Lys-455 a nd of His-460 with Glu gave mutant LHRs that failed to localize or fol d properly at the cell surface as evidenced by the lack of significant binding to intact cells, although hCG binding could be detected in br oken cell preparations, and a neighboring Arg-459 --> Glu replacement had no apparent effect on receptor trafficking, hCG binding or hCG-med iated cAMP-production. A reversal mutant in ICL II in which Glu-441, a t the boundary of transmembrane helix III and ICL II, and His-460, at the interface between ICL II and transmembrane helix IV, were intercha nged, exhibited hCG binding to intact cells, but the maximal cAMP leve l at high concentrations of ligand was less than that obtained with CO S-7 cells transfected with wild-type LHR. The total number of cell sur face receptors determined with the reversal mutant was less than that found with wild-type LHR. This difference, however, is not believed to be responsible for the reduced signaling, since maximal cAMP response s to hCG were obtained with comparable receptor densities of wild-type and various mutant LHRs. Other single replacements in ICL I, Lys-368 --> Glu and to Gin, and in ICL III, Arg-526 --> Glu and Tyr-528 --> Se r, resulted in mutant LHRs with characteristics of wild-type LHR in tr afficking, hCG binding and hCG-mediated cAMP production. These finding s suggest an important functional role of several amino acid residues in ICL II of LHR. (C) 1997 Elsevier Science Ireland Ltd.