Juxtamembrane regions in the third intracellular loop of the thyrotropin-releasing hormone receptor type 1 are important for coupling to Gq

Juxtamembrane residues in the putative third intracellular (I3) loops of a number of G protein-coupled receptors (GPCRs) have been shown to be importa nt for coupling to G proteins. According to standard hydropathy analysis, t he I3 loop of the mouse TRH receptor type 1 (mTRH-R1) is composed of 51 ami no acids from position-213 to position-263. We constructed deletion and sit e-specific I3 loop TRH-R mutants and studied their binding and TRH-stimulat ed signaling activities. As expected, the effects of these mutations on TRH binding were small (less than 5-fold decreases in affinity). No effect on TRH-stimulated signaling activity was found in a mutant receptor in which t he I3 loop was shortened to 16 amino acids by deleting residues from Asp-22 6 to Ser-260. In contrast, mutants with deletions from Asp-222 to Ser-260 o r from Asp-226 to Gln-263 exhibited reduced TRH-stimulated signaling. In th e region near transmembrane helix 6, single site-specific substitution of e ither Arg-261 or Lys-262 by neutral glutamine had little effect on signalin g, but mutant TRH-Rs that were substituted by glutamine at both basic resid ues exhibited reduced TRH-stimulated activity. The reduced signaling activi ty of this doubly substituted mutant was reversed by over expressing the al pha subunit of Gq. These data demonstrate that the juxtamembrane regions in the TRH-R I3 loop are important for coupling to Gq.