A highly conserved tryptophan residue in the fourth transmembrane domain of the A1 adenosine receptor is essential for ligand binding but not receptor homodimerization

Tokiko Suzuki, Kazunori Namba, Ryosuke Yamagishi, Hiroki Kaneko, Tatsuya Haga, Hiroyasu Nakata

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Dimerization between G protein-coupled receptors (GPCRs) is a clearly established phenomenon. However, limited information is currently available on the interface essential for this process. Based on structural comparisons and sequence homology between rhodopsin and A1 adenosine receptor (A 1R), we initially hypothesized that four residues in transmembrane (TM) 4 and TM5 are involved in A1R homodimerization. Accordingly, these residues were substituted with Ala by site-directed mutagenesis. Interestingly, the mutant protein displayed no significant decrease in homodimer formation compared with wild-type A1R, as evident from coimmunoprecipitation and BRET2 analyses (improved bioluminescence resonance energy transfer system offered by Perkin-Elmer Life Sciences), but lost ligand binding activity almost completely. Further studies disclosed that this effect was derived from the mutation of one particular residue, Trp132, which is highly conserved among many GPCRs. Confocal immunofluorescence and cell-surface biotinylation studies revealed that the mutant receptors localized normally at transfected cell membranes, signifying that loss of ligand binding was not because of defective cellular trafficking. Molecular modeling of the A1R-ligand complex disclosed that Trp132 interacted with several residues located in TM3 and TM5 that stabilized agonist binding. Thus, loss of interactions of Trp with these residues may, in turn, disrupt binding to agonists. Our study provides strong evidence of the essential role of the highly conserved Trp132 in TM4 of adenosine receptors.

Original languageEnglish
Pages (from-to)1352-1362
Number of pages11
JournalJournal of Neurochemistry
Volume110
Issue number4
DOIs
Publication statusPublished - 2009 Aug 1

Keywords

  • Adenosine receptor
  • Agonist binding model
  • G protein-coupled receptors
  • Oligomerization
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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