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Structural Determinants for G Protein Activation and Selectivity in the Second Intracellular Loop of the Thyrotropin Receptor

Third Medical Department, University of Leipzig (S.N., M.C., R.P.), D-04103 Leipzig, Germany; and Institute for Molecular Pharmacology (G.K.), D-13125 Berlin, Germany

Address all correspondence and requests for reprints to: Dr. Ralf Paschke, Third Medical Department, University of Leipzig, Ph. Rosenthal Strasse 27, 04103 Leipzig, Germany. E-mail: ralf.paschke{at}medizin.uni-leipzig.

The TSH receptor (TSHR) activates mainly two signal transduction pathways, cAMP production and phosphoinositide turnover, mediated by G_s and G_q coupling, respectively. Several activating deletion and point mutations within intracellular loop 3 (ICL3) and the adjacent portion of transmembrane domain 6 (TM6) support a direct G protein activation by this receptor domain. The ICL3, however, is predicted by modeling to interact with other receptor domains, primarily ICL2, to form a pocket for G protein binding and to allow optimum interaction. Systematic mutagenesis was used to identify important sites within ICL2 and potential interactions between ICL2 and ICL3 of the TSHR required for G protein coupling. Deletions of four or five residues and their corresponding multiple alanine substitutions were introduced into ICL2. Residues I523-D530, comprising mainly the N-terminal half of ICL2, appeared to be critical for G_s- and G_q-mediated signaling. A single alanine substitution screening within ICL2 revealed hydrophobic residue M527 in particular and, to lesser extents, F525, R528, L529, and D530 as residues that selectively abolished or strongly impaired G_q activation. Molecular modeling suggests that F525 interacts with ICL3. To test this hypothesis, ICL2/ICL3 double mutants introducing strong complementary properties were constructed and tested for functional rescue of G_q-mediated signaling. Our results indicate that ICL2 interacts with ICL3 in close vicinity to F525 and T607, suggesting a conformational cooperation between ICL2 and ICL3 during G_q activation by TSHR.

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