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Modelling the P2Y purinoceptor using rhodopsin as template.

ABSTRACT: The P2Y1 purinoceptor cloned from chick brain (Webb, T. et al (1993) FEBS Lett., 324, 219-225) is a 362 amino acid, 41 kDa protein. To locate residues tentatively involved in ligand recognition a molecular model of the P2Y purinoceptor has been constructed. The model was based on the primary sequence and structural homology with the G-protein coupled photoreceptor rhodopsin, in analogy to the method proposed by Ballesteros and Weinstein ((1995) Meth. Neurosci. 25, 366-428). Transmembrane helices were constructed from the amino acid sequence, minimized individually, and positioned in a helical bundle. The helical bundle was then minimized using the Amber forcefield in Discover (BIOSYM Technologies) to obtain the final model. Several residues that have been shown to be critical in ligand binding in other GPCRs are conserved in the P2Y1 purinoceptor. According to our model the side chains of these conserved residues are facing the internal cleft in which ligand binding likely occurs. The model also suggests four basic residues (H121 in TM3, H266 and K269 in TM6 and R299 in TM7) near the extracellular surface that might be involved in ligand binding. These basic residues might be essential in coordinating the triphosphate chain of the endogenous ligand adenosine 5'-triphosphate (ATP). Potential binding sites for agonists have been explored by docking several derivatives (including newly synthesized N6-derivatives) into the model. The N6-phenylethyl substituent is tolerated pharmacologically, and in our model this substituent occupies a region predominantly defined by aromatic residues such as F51 (TM1), Y100 (TM2) and F120 (TM3). The dimethylated analogue of ATP, N6,N6-dimethyl-adenosine 5'-triphosphate, is less well tolerated pharmacologically, and our model predicts that the attenuated activity is due to interference with hydrogen bonding capacity to Q296 (TM7).

SUBMITTER: Van Rhee AM

PROVIDER: S-EPMC3448275 | biostudies-literature | 1995 Nov

REPOSITORIES: biostudies-literature

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Modelling the P2Y purinoceptor using rhodopsin as template.

Van Rhee A M AM Fischer B B Van Galen P J PJ Jacobson K A KA

Drug design and discovery 19951101 2

The P2Y1 purinoceptor cloned from chick brain (Webb, T. et al (1993) FEBS Lett., 324, 219-225) is a 362 amino acid, 41 kDa protein. To locate residues tentatively involved in ligand recognition a molecular model of the P2Y purinoceptor has been constructed. The model was based on the primary sequence and structural homology with the G-protein coupled photoreceptor rhodopsin, in analogy to the method proposed by Ballesteros and Weinstein ((1995) Meth. Neurosci. 25, 366-428). Transmembrane helices ...[more]

PMID: 8872457

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Project description:Activation of G Protein-Coupled Receptors (GPCRs) is an allosteric mechanism triggered by ligand binding and resulting in conformational changes transduced by the transmembrane domain. Models of the activated forms of GPCRs have become increasingly necessary for the development of a clear understanding of signal propagation into the cell. Experimental evidence points to a multiplicity of conformations related to the activation of the receptor, rendered important physiologically by the suggestion that different conformations may be responsible for coupling to different signaling pathways. In contrast to the inactive state of rhodopsin (RHO) for which several high quality X-ray structures are available, the structure-related information for the active states of rhodopsin and all other GPCRs is indirect. We have collected and stored such information in a repository we maintain for activation-specific structural data available for rhodopsin-like GPCRs, http://www.physiology.med.cornell.edu/GPCRactivation/gpcrindex.html . Using these data as structural constraints, we have applied Simulated Annealing Molecular Dynamics to construct a number of different active state models of RHO starting from the known inactive structure. The common features of the models indicate that TM3 and TM5 play an important role in activation, in addition to the well-established rearrangement of TM6. Some of the structural changes observed in these models occur in regions that were not involved in the constraints, and have not been previously tested experimentally; they emerge as interesting candidates for further experimental exploration of the conformational space of activated GPCRs. We show that none of the normal modes calculated from the inactive structure has a dominant contribution along the path of conformational rearrangement from inactive to the active forms of RHO in the models. This result may differentiate rhodopsin from other GPCRs, and the reasons for this difference are discussed in the context of the structural properties and the physiological function of the protein.

Project description:prolonged P2Y-receptor signalling can cause vasoconstriction leading to hypertension, vascular smooth muscle hypertrophy, and hyperplasia. G protein-coupled receptor signalling is negatively regulated by G protein-coupled receptor kinases (GRKs) and arrestin proteins, preventing prolonged or inappropriate signalling. This study investigates whether GRKs and arrestins regulate uridine 5'-triphosphate (UTP)-stimulated contractile signalling in adult Wistar rat mesenteric arterial smooth muscle cells (MSMCs).mesenteric arteries contracted in response to UTP challenge: When an EC(50) UTP concentration (30 µM, 5 min) was added 5 min before (R(1)) and after (R(2)) the addition of a maximal UTP concentration (R(max): 100 µM, 5 min), R(2) responses were decreased relative to R(1), indicating desensitization. UTP-induced P2Y-receptor desensitization of phospholipase C signalling was studied in isolated MSMCs transfected with an inositol 1,4,5-trisphosphate biosensor and/or loaded with Ca(2+)-sensitive dyes. A similar protocol (R(1)/R(2) = 10 µM; R(max) = 100 µM, applied for 30 s) revealed markedly reduced R(2) when compared with R(1) responses. MSMCs were transfected with dominant-negative GRKs or siRNAs targeting specific GRK/arrestins to probe their respective roles in P2Y-receptor desensitization. GRK2 inhibition, but not GRK3, GRK5, or GRK6, attenuated P2Y-receptor desensitization. siRNA-mediated knockdown of arrestin2 attenuated UTP-stimulated P2Y-receptor desensitization, whereas arrestin3 depletion did not. Specific siRNA knockdown of the P2Y(2)-receptor almost completely abolished UTP-stimulated IP(3)/Ca(2+) signalling, strongly suggesting that our study is specifically characterizing this purinoceptor subtype.these new data highlight roles of GRK2 and arrestin2 as important regulators of UTP-stimulated P2Y(2)-receptor responsiveness in resistance arteries, emphasizing their potential importance in regulating vasoconstrictor signalling pathways implicated in vascular disease.

Project description:BACKGROUND:Premenopausal women have a lower risk of hypertension compared to age-matched men and postmenopausal women. P2Y2 and P2Y4 purinoceptor can be considered potential contributors to hypertension due to their emerging roles in regulating renal tubular Na+ transport. Activation of these receptors inhibits epithelial Na+ channel activity (ENaC) via a phospholipase C (PLC)-dependent pathway resulting in natriuresis. We recently reported that activation of P2Y2 and P2Y4 receptors in the renal medulla by UTP promotes natriuresis in male and ovariectomized (OVX) rats, but not in ovary-intact females. This led us to hypothesize that ovary-intact females have greater basal renal medullary activity of P2 (P2Y2 and P2Y4) receptors regulating Na+ excretion compared to male and OVX rats. METHODS:To test our hypothesis, we determined (i) the effect of inhibiting medullary P2 receptors by suramin (750??g/kg/min) on urinary Na+ excretion in anesthetized male, ovary-intact female, and OVX Sprague Dawley rats, (ii) mRNA expression and protein abundance of P2Y2 and P2Y4 receptors, and (iii) mRNA expression of their downstream effectors (PLC-1? and ENaC?) in renal inner medullary tissues obtained from these three groups. We also subjected cultured mouse inner medullary collecting duct cells (segment 3, mIMCD3) to different concentrations of 17ß-estradiol (E2, 0, 10, 100, and 1000?nM) to test whether E2 increases mRNA expression of P2Y2 and P2Y4 receptors. RESULTS:Acute P2 inhibition attenuated urinary Na+ excretion in ovary-intact females, but not in male or OVX rats. We found that P2Y2 and P2Y4 mRNA expression was higher in the inner medulla from females compared to males or OVX. Inner medullary lysates showed that ovary-intact females have higher P2Y2 receptor protein abundance, compared to males; however, OVX did not eliminate this sex difference. We also found that E2 dose-dependently upregulated P2Y2 and P2Y4 mRNA expression in mIMCD3. CONCLUSION:These data suggest that ovary-intact females have enhanced P2Y2 and P2Y4-dependent regulation of Na+ handling in the renal medulla, compared to male and OVX rats. We speculate that the P2 pathway contributes to facilitated renal Na+ handling in premenopausal females.

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Modelling the P2Y purinoceptor using rhodopsin as template.

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Modelling the P2Y purinoceptor using rhodopsin as template.

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