Colonic crypt cells, depending on regulatory influences such as neurotransmitters and hormones, can absorb or secrete NaCl. The large changes in absolute rates of transport, multiplicity of regulatory pathways, and complete reversal of vectorial transport in these cells pose intriguing questions. Some of these questions have been examined recently;most answers came as a complete surprise.

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.

The P2Y11-R (P2Y11 receptor) is a less explored drug target. We computed an hP2Y11-R (human P2Y11) homology model with two templates, bovine-rhodopsin (2.6 A resolution; 1 A=0.1 nm) and a hP2Y1-ATP complex model. The hP2Y11-R model was refined using molecular dynamics calculations and validated by virtual screening methods, with an enrichment factor of 5. Furthermore, mutational analyses of Arg106, Glu186, Arg268, Arg307 and Ala313 confirmed the adequacy of our hP2Y11-R model and the computed ligand recognition mode. The E186A and R268A mutants reduced the potency of ATP by one and three orders of magnitude respectively. The R106A and R307A mutants were functionally inactive. We propose that residues Arg106, Arg268, Arg307 and Glu186 are involved in ionic interactions with the phosphate moiety of ATP. Arg307 is possibly also H-bonded to N6 of ATP via the backbone carbonyl. Activity of ATP at the F109I mutant revealed that the proposed p-stacking of Phe109 with the adenine ring is a minor interaction. The mutation A313N, which is part of a hydrophobic pocket in the vicinity of the ATP C-2 position, partially explains the high activity of 2-MeS-ATP at P2Y1-R as compared with the negligible activity at the P2Y11-R. Inactivity of ATP at the Y261A mutant implies that Tyr261 acts as a molecular switch, as in other G-protein-coupled receptors. Moreover, analysis of cAMP responses seen with the mutants showed that the efficacy of coupling of the P2Y11-R with Gs is more variable than coupling with Gq. Our model also indicates that Ser206 forms an H-bond with Pgamma (the gamma-phosphate of the triphosphate chain of ATP) and Met310 interacts with the adenine moiety.

Background and purpose: We explored the stereoselective activation of the P2Y 11 receptor, stably expressed and tagged with GFP, in 1321N1 cells, in comparison to its closest homologue, the P2Y 1 receptor. Experimental approach: The potency of several chiral ATP analogues was determined by measuring increases in intracellular calcium concentration ([Ca 2 þ ] i ). In a series of ATP-a-B and ATP-a-S analogues, a non-bridging oxygen atom of P a was substituted by BH 3 or sulphur, respectively, introducing a chiral center at P a . The pairs of diastereoisomers (A and B isomers) were each applied as purified compounds. Key results: The (B) isomers (ATP-a-B Sp isomers and ATP-a-S Rp isomers) of all derivatives tested were more potent at the P2Y 11 receptor than the corresponding (A) isomers (ATP-a-B Rp isomers and ATP-a-S Sp isomers) and the parent compounds. This characteristic of the P2Y 11 receptor is opposite to the behaviour of the same diastereoisomers at the P2Y 1 receptor, at which the (A) isomers are more active. Conclusions and implications:The distinctly opposite diastereoselective activity of ATP derivatives at the P2Y 11 and the P2Y 1 receptor will allow the deciphering of structural differences of the ligand recognition sites between these receptor subtypes and may aid in the development of subtype-selective agonists. Moreover, ATP-a-B diastereoisomers are not active at the P2Y 2 receptor. Thus, they are compounds suitable for distinguishing the functional contribution of the two ATP-activated P2Y receptors, the P2Y 2 and P2Y 11 receptor, in physiological or pathophysiological responses of cells. British Journal of Pharmacology

Background and purpose: The P2Y 11 receptor, a member of the group of metabotropic nucleotide receptors, shows a stereospecific ligand recognition of P a -substituted ATP derivatives (ATP-a-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-a-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 a -substituted ATP derivatives is largely determined by the basic residue Arg268 in TM6. This will allow the design of receptor-subtype selective ligands.

N1,N 6 -ethenoadenine, ε-A, nucleos(t)ides have been previously applied as fluorescent probes in numerous biochemical systems. However, these ε-A analogues lack the H-bonding capability of adenine. To improve the fluorescence characteristics while preserving the H-bonding pattern required for molecular recognition, we designed a novel probe: N 2 ,N3-etheno-adenosine, (N 2 ,N3-ε-A). Here, we describe four novel syntheses of the target ε-nucleoside and related analogues.These methods are short, facile, and provide the product regiospecifically. In addition, we report the absorption and emission spectra of N 2 ,N3-ε-A and the dependence of the spectral features on the pH and polarity of the medium. Specifically, maximum emission of N 2 ,N3-ε-A in water is observed at 420 nm (ϕ=0.03, excitation at 290 nm). The biochemical relevance of the new probe was evaluated with respect to the P2Y 1 receptor and NTPDases 1 and 2. N 2 ,N3-ε-ATP was found to be almost equipotent with ATP at the P2Y 1 receptor and was hydrolyzed by NTPDases 1 and 2at about 80% of the rate of ATP. Furthermore, protein binding does not seem to shift the fluorescence of N 2 ,N3-ε-ATP. Based on the fluorescence and full recognition by ATP-binding proteins, we propose N 2 ,N3-ε-ATP and related nucleo(s)tides as unique probes for the investigation of adenine nucleo(s)tide-binding proteins as well as for other biochemical applications.