The TSH receptor (TSHR) activates mainly two signal transduction pathways, cAMP production and phosphoinositide turnover, mediated by Gs and Gq 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 Gs- and Gq-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 Gq 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 Gq-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 Gq activation by TSHR.