Identification of transmembrane domain 6 & 7 residues that contribute to the binding pocket of the urotensin II receptor

Biochem Pharmacol. 2009 Apr 15;77(8):1374-82. doi: 10.1016/j.bcp.2009.01.013. Epub 2009 Jan 29.


Urotensin II (U-II), a cyclic undecapeptide, is the natural ligand of the urotensin II (UT) receptor, a G protein-coupled receptor. In the present study, we used the substituted-cysteine accessibility method to identify specific residues in transmembrane domains (TMDs) six and seven of the rat urotensin II receptor (rUT) that contribute to the formation of the binding pocket of the receptor. Each residue in the R256(6.32)-Q283(6.59) fragment of TMD6 and the A295(7.31)-T321(7.57) fragment of TMD7 was mutated, individually, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the positively charged methanethiosulfonate-ethylammonium (MTSEA) or the negatively charged methanethiosulfonate-ethylsulfonate (MTSES) sulfhydryl-specific alkylating agents. MTSEA treatment resulted in a significant reduction in the binding of TMD6 mutants F268C(6.44) and W278C(6.54) and TMD7 mutants L298C(7.34), T302C(7.38), and T303C(7.39) to (125)I-U-II. MTSES treatment resulted in a significant reduction in the binding of two additional mutants, namely L282C(6.58) in TMD6 and Y300C(7.36) in TMD7. These results suggest that specific residues orient themselves within the water-accessible binding pocket of the rUT receptor. This approach, which allowed us to identify key determinants in TMD6 and TMD7 that contribute to the UT receptor binding pocket, enabled us to further refine our homology-based model of how U-II interacts with its cognate receptor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylating Agents / pharmacology
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Cysteine / genetics
  • Cysteine / metabolism*
  • Ethyl Methanesulfonate / analogs & derivatives
  • Ethyl Methanesulfonate / pharmacology
  • Ligands
  • Mesylates / pharmacology
  • Models, Molecular
  • Mutation*
  • Polymerase Chain Reaction
  • Protein Binding
  • Protein Structure, Tertiary
  • Radioligand Assay
  • Rats
  • Receptors, G-Protein-Coupled / chemistry
  • Receptors, G-Protein-Coupled / genetics
  • Receptors, G-Protein-Coupled / metabolism*
  • Transfection
  • Urotensins / pharmacology


  • Alkylating Agents
  • Ligands
  • Mesylates
  • Receptors, G-Protein-Coupled
  • Urotensins
  • Uts2r protein, rat
  • methanethiosulfonate ethylammonium
  • (2-sulfonatoethyl)methanethiosulfonate
  • urotensin II
  • Ethyl Methanesulfonate
  • Cysteine