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Premium A highly conserved tryptophan residue in the fourth transmembrane domain of the A 1 adenosine receptor is essential for ligand binding but not receptor homodimerization
Author(s)
Suzuki Tokiko,
Namba Kazunori,
Yamagishi Ryosuke,
Kaneko Hiroki,
Haga Tatsuya,
Nakata Hiroyasu
Publication year2009
Publication title
journal of neurochemistry
Resource typeJournals
PublisherBlackwell Publishing Ltd
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 A 1 adenosine receptor (A 1 R), we initially hypothesized that four residues in transmembrane (TM) 4 and TM5 are involved in A 1 R 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 A 1 R, as evident from coimmunoprecipitation and BRET 2 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 A 1 R‐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.
Subject(s)adenosine receptor , agonist , binding site , biochemistry , biology , biophysics , chemistry , fluorescence , förster resonance energy transfer , g protein coupled receptor , gene , microbiology and biotechnology , mutagenesis , mutant , physics , quantum mechanics , receptor , retinal , rhodopsin , transmembrane domain
Language(s)English
SCImago Journal Rank1.75
H-Index229
eISSN1471-4159
pISSN0022-3042
DOI10.1111/j.1471-4159.2009.06227.x

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