PARENT SESSION
MECHANISMS OF HORMONE ACTION

Tuesday, August 3, 2004
10:30 AM–12:30 PM
Buchanan Courtyard

(333) ROLE OF INTRACELLULAR LOOP 2 IN THE ACTIVATION OF THE HUMAN LUTEINIZING HORMONE RECEPTOR.

Angelova, Krassimira ¹, Fanelli, Francesca², Narayan, Prema¹, Puett, David¹, ¹ University of Georgia, Athens, GA² University of Modena and Reggio Emilia, Modena, Italy

ABSTRACT- The LH receptor (LHR) contains a large extracellular domain responsible for agonist binding and a typical GPCR membrane component. Activation of the receptor is associated with conformational changes that are propagated to the cytosolic side where G protein coupling occurs. Our recent molecular model of constitutive LHR activation suggests that the amino acid residues at the junction of transmembrane helices and intracellular loops (ICL), e.g. helix 3/ICL2 and helix 5/ICL3, are important for G protein activation. The aim of this study was to determine the residues in ICL2 of human LHR that are critical for receptor function. Individual amino acid residues were analyzed by Ala scanning mutagenesis with the mutant LHRs being transiently expressed in HEK 293 cells and characterized for cell surface binding and cAMP signaling. Of 22 single receptor mutants characterized, only the E463A mutant did not express and five mutants expressed poorly (below 30% of wt Bmax). Ligand binding and basal cAMP levels of the mutants were similar to that of the wt LHR and all of them were responsive to hCG. However, the coupling efficiencies of mutants I460A, T461A, R464A, H466A, I468A, I472A and L478A were lower, and, interestingly, W465A and K477A coupled more efficiently than the wt LHR. Three double mutants, pairing the constitutively active mutant, D578H in TM6, with each of three mutants with lower coupling efficiency, T461A, R464A, and I468A, showed elevated basal cAMP level relative to wt LHR but only R464A/D578H was responsive to hCG, although with lower coupling efficiency than wt LHR. T461A/I468A, pairing the two low coupling mutants, showed wt-like basal cAMP and was marginally responsive to ligand.The results of engineered mutagenesis, coupled with homology modeling, suggest different roles of the amino acid residues in receptor function. In particular, of the mutated residues in helix 3, only those that face helices 5 and 6, i.e. I460, T461, R464 and I468, are required for hCG stimulation. Many of the others are likely to play an intramolecular structural role. Supported by NIH DK33973.

KEY WORDS: Mutagenesis, Molecular modeling, LH receptor activation, Coupling efficiency