2 MUTATIONS OF THE LUTROPIN CHORIOGONADOTROPIN RECEPTOR THAT IMPAIR SIGNAL-TRANSDUCTION ALSO INTERFERE WITH RECEPTOR-MEDIATED ENDOCYTOSIS/

The experiments presented herein were designed to probe a potential ro le for the activation of the LH/CG receptor (LHR) on the receptor-medi ated endocytosis of human CG (hCG). Two mutants of the rat LHR (rLHR) that bind the hormone with high affinity but are deficient in signal t ransduction were prepared by mutating highly conserved residues that h ave been previously shown to be important in signal transduction in ot her members of the G protein-coupled receptor family. Mutation of a hi ghly conserved aspartic acid in the second transmembrane domain of the rLHR (designated rLHR-D383N) does not affect hCG binding but impairs signal transduction. When compared with cells expressing an equivalent density of wild type rLHR (rLHR-wt), concentration-response curves fo r the hCG-stimulated cAMP accumulation in cells expressing rLRH-D383N are characterized by an 18-fold increase in the EC(50) but no change i n the maximal response. Cells expressing rLHR-D383N also display a 4- to 5-fold increase in the half-life of internalization of hCG. Mutatio n of a highly conserved arginine in the second intracellular loop of t he rLHR (designated rLHR-R442H) also does not affect hCG binding but i mpairs signal transduction. When compared with cells expressing an equ ivalent density of rLHR-wt, concentration-response curves for the hCG- stimulated cAMP accumulation in cells expressing rLHR-R442H are charac terized by a 7-fold increase in the EC(50) and a 6- to 10-fold decreas e in the maximal response. Cells expressing rLHR-R442H also display a 1.5- to 2-fold increase in the half-life of internalization of hCG. Th ese results, together with the finding that an antagonist of hCG is in ternalized more slowly than hCG, suggest that the activation of the LH R is needed for the efficient endocytosis of the bound hCG.