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Diastereoselectivity of the P2Y11 nucleotide receptor: mutational analysis.

ABSTRACT: BACKGROUND AND PURPOSE: The P2Y(11) receptor, a member of the group of metabotropic nucleotide receptors, shows a stereospecific ligand recognition of P(alpha)-substituted ATP derivatives (ATP-alpha-S isomers). These compounds are suitable candidates for the development of selective P2Y(11) receptor agonists that might be used as immune modulators. We have analysed the binding mode of ATP at the P2Y(11) receptor by molecular modeling and site-directed mutagenesis. Based on our recent findings, we decided to decipher the molecular determinants of stereoselective recognition at the P2Y(11) receptor. EXPERIMENTAL APPROACH: Two amino acid residues [Glu186 in the extracellular loop 2 and Arg268 in the transmembrane domain 6 (TM6)], which are part of the nucleotide-binding pocket, were selected and studied by mutational analyses. We expected these residues to be involved in determining the stereospecificity of the P2Y(11) receptor. KEY RESULTS: After mutation of Arg268 to alanine or glutamine, the stereospecific recognition of the ATP-alpha-S isomers at the P2Y(11) receptor was lost. In contrast, at the Glu186Ala receptor mutant, the stereoselective differentiation between these isomers was increased. On the Arg268Gln/Glu186Ala double mutant we observed no further effect, except for additivity in the decrease in potency of both isomers, as compared with the single-point mutants. CONCLUSIONS AND IMPLICATIONS: Our results show that the stereospecificity of the P2Y(11) receptor for P(alpha)-substituted ATP derivatives is largely determined by the basic residue Arg268 in TM6. This will allow the design of receptor-subtype selective ligands.

SUBMITTER: Ecke D

PROVIDER: S-EPMC2607205 | biostudies-other | 2008 Dec

REPOSITORIES: biostudies-other

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Project description:Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse ?2-adrenergic-receptor (m?2AR), robustly traffics to the plasma membrane. We set out to characterize m?2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of m?2AR using a Green Fluorescent Protein-tagged m?2AR (m?2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known m?2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of m?2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of m?2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three m?2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower m?2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs.

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Diastereoselectivity of the P2Y11 nucleotide receptor: mutational analysis.

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