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 important for coupling to G proteins. According to standard hydropathy analysis, the I3 loop of the mouse TRH receptor type 1 (mTRH-R1) is composed of 51 amino acids from position-213 to position-263. We constructed deletion and site-specific I3 loop TRH-R mutants and studied their binding and TRH-stimulated 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 the I3 loop was shortened to 16 amino acids by deleting residues from Asp-226 to Ser-260. In contrast, mutants with deletions from Asp-222 to Ser-260 or from Asp-226 to Gln-263 exhibited reduced TRH-stimulated signaling. In the region near transmembrane helix 6, single site-specific substitution of either Arg-261 or Lys-262 by neutral glutamine had little effect on signaling, but mutant TRH-Rs that were substituted by glutamine at both basic residues exhibited reduced TRH-stimulated activity. The reduced signaling activity of this doubly substituted mutant was reversed by over expressing the a subunit of Gq. These data demonstrate that the juxtamembrane regions in the TRH-R I3 loop are important for coupling to Gq.
Buck
F
F
Institut für Zellbiochemie und Klinische Neurobiologie, Universitäts-Krankenhaus Eppendorf, Hamburg, Germany.
Wang
W
W
Harder
S
S
Brathwaite
C
C
Bruhn
T O
TO
Gershengorn
M C
MC
D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D005260:Female; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D008024:Ligands; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009865:Oocytes; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014982:Xenopus laevis
D008024:Ligands; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins