Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.     

P2Y11 receptors activate adenylyl cyclase and contribute to nucleotide-promoted cAMP formation in MDCK-D(1) cells. A mechanism for nucleotide-mediated autocrine-paracrine regulation.

Extracellular nucleotides activate P2Y receptors, thereby increasing cAMP formation in Madin-Darby canine kidney (MDCK-D(1)) cells, which express P2Y(1), P2Y(2), and P2Y(11) receptors (Post, S. R., Rump, L. C., Zambon, A., Hughes, R. J., Buda, M. D., Jacobson, J. P., Kao, C. C., and Insel, P. A. (1998) J. Biol. Chem. 273, 23093-23097). The cyclooxygenase inhibitor indomethacin (indo) eliminates UTP-promoted cAMP formation (i.e. via P2Y(2) receptors) but only partially blocks ATP-promoted cAMP formation. The latter response is completely blocked by the nonselective P2Y receptor antagonist suramin. We have sought to identify the mechanism for this P2Y receptor-mediated, indo-resistant cAMP formation. The agonist rank order potencies for cAMP formation were: ADP beta S > or = MT-ADP > 2-MT-ATP > ADP, ATP, ATP gamma S > UTP, AMP, adenosine. We found a similar rank order in MDCK-D(1) cells overexpressing cloned green fluorescent protein-tagged P2Y(11) receptors, but the potency of the agonists was enhanced, consistent with a P2Y(11) receptor-mediated effect. cAMP generation by the P2Y(1) and P2Y(11) receptor agonist ADP beta S was not inhibited by several P2Y(1)-selective antagonists (PPADS, A2P5P, and MRS 2179). Forskolin synergistically enhanced cAMP generation in response to ADP beta S or PGE(2), implying that, like PGE(2), ADP beta S activates adenylyl cyclase via G(s), a conclusion supported by results showing ADP beta S and MT-ADP promoted activation of adenylyl cyclase activity in MDCK-D(1) membranes. We conclude that nucleotide-promoted, indo-resistant cAMP formation in MDCK-D(1) cells occurs via G(s)-linked P2Y(11) receptors. These data describing adenylyl cyclase activity via endogenous P2Y(11) receptors define a mechanism by which released nucleotides can increase cAMP in MDCK-D(1) and other P2Y(11)-containing cells.     

Benzoyl ATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation

The effects of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) on intracellular Ca2+ mobilization and cyclic AMP accumulation were investigated using rat brain capillary endothelial cells which express an endogenous P2Y1 receptor, human platelets which are known to express a P2Y1 receptor, and Jurkat cells stably transfected with the human P2Y1 receptor. In endothelial cells, BzATP was a competitive inhibitor of 2-methylthio ADP (2-MeSADP) and ADP induced [Ca2+]i responses (Ki = 4.7 microM) and reversed the inhibition by ADP of adenylyl cyclase (Ki = 13 microM). In human platelets, BzATP inhibited ADP-induced aggregation (Ki = 5 microM), mobilization of intracellular Ca2+ stores (Ki = 6.3 microM), and inhibition of adenylyl cyclase. In P2Y1-Jurkat cells, BzATP inhibited ADP and 2-MeSADP-induced [Ca2+]i responses (Ki = 2.5 microM). It was concluded that BzATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. In contrast to other P2Y1 receptor antagonists (A2P5P and A3P5P) which inhibit only ADP-induced Ca2+ mobilization, BzATP inhibits both the Ca2+- and the cAMP-dependent intracellular signaling pathways of ADP. These results provide further evidence that P2Y1 receptors contribute to platelet ADP responses.     

Modulation of ion channel function by P2Y receptors

P2Y receptors are classified as P2 purinergic receptors that belong to the superfamily of G-protein coupled receptors. They are distinguishable from P1 (adenosine) receptors in that they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species. Eight functional subtypes have been characterized. Nucleotide binding produces activation of specific G-proteins that in turn regulate the function of membrane bound enzymes including phospholipase C and adenylyl cyclase. Certain P2Y receptor subtypes possess a PDZ domain located at the end of the C-terminal region of the receptor. PDZ domains have been established as sites for protein-protein interaction, thus providing a possible mechanism for receptor modulation of membrane protein function independent of G-protein activation. In this review we discuss recent findings that suggest that P2Y receptors can modulate the function of ion channels through multiple protein-protein interactions at the plasma membrane that do not directly involve G-protein activation.     

Cross-talk between the ATP and ADP nucleotide receptor signalling pathways in glioma C6 cells

In this review we summarize the present status of our knowledge on the enzymes involved in the extracellular metabolism of nucleotides and the receptors involved in nucleotide signalling. We focus on the mechanism of the ATP and ADP signalling pathways in glioma C6, representative of the type of nonexcitable cells. In these cells, ATP acts on the P2Y(2) receptor coupled to phospholipase C, whereas ADP on two distinct P2Y receptors: P2Y(1) and P2Y(12). The former is linked to phospholipase C and the latter is negatively coupled to adenylyl cyclase. The possible cross-talk between the ATP-, ADP- and adenosine-induced pathways, leading to simultaneous regulation of inositol 1,4,5-trisphosphate and cAMP mediated signalling, is discussed.     

Characterization and channel coupling of the P2Y(12) nucleotide receptor of brain capillary endothelial cells

Rat brain capillary endothelial (B10) cells express an unidentified nucleotide receptor linked to adenylyl cyclase inhibition. We show that this receptor in B10 cells is identical in sequence to the P2Y(12) ADP receptor ("P2Y(T)") of platelets. When expressed heterologously, 2-methylthio-ADP (2-MeSADP; EC(50), 2 nm), ADP, and adenosine 5'-O-(2-thio)diphosphate were agonists of cAMP decrease, and 2-propylthio-D-beta,gamma-difluoromethylene-ATP was a competitive antagonist (K(B), 28 nm), as in platelets. However, 2-methylthio-ATP (2-MeSATP) (EC(50), 0.4 nm), ATP (1.9 microm), and 2-chloro-ATP (190 nm), antagonists in the platelet, were also agonists. 2-MeSADP activated (EC(50), 0.1 nm) GIRK1/GIRK2 inward rectifier K(+) channels when co-expressed with P2Y(12) receptors in sympathetic neurons. Surprisingly, P2Y(1) receptors expressed likewise gave that response; however, a full inactivation followed, absent with P2Y(12) receptors. A new P2Y(12)-mediated transduction was found, the closing of native N-type Ca(2+) channels; again both 2-MeSATP and 2-MeSADP are agonists (EC(50), 0.04 and 0.1 nm, respectively). That action, like their cAMP response, was pertussis toxin-sensitive. The Ca(2+) channel inhibition and K(+) channel activation are mediated by beta gamma subunit release from a heterotrimeric G-protein. G alpha subunit types in B10 cells were also identified. The presence in the brain capillary endothelial cell of the P2Y(12) receptor is a significant extension of its functional range.     

The P2Y11 receptor mediates the ATP-induced maturation of human monocyte-derived dendritic cells

Recently, it has been shown that ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-alpha and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5'-O-(3-thiotriphosphate) = 2'- and 3'-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.     

Distinctive features of Trk neurotrophin receptor transactivation by G protein-coupled receptors.

Ligands for G protein-coupled receptors (GPCR) are capable of activating mitogenic receptor tyrosine kinases, in addition to the mitogen-activated protein (MAP) kinase signaling pathway and classic G protein-dependent signaling pathways involving adenylyl cyclase and phospholipase. For example, receptors for epidermal growth factor (EGF), insulin-like growth-1 and platelet-derived growth factor and can be transactivated through G protein-coupled receptors. Neurotrophins, such as NGF, BDNF and NT-3 also utilize receptor tyrosine kinases, namely TrkA, TrkB and TrkC. Recently, it has been shown that activation of Trk receptor tyrosine kinases can also occur via a G protein-coupled receptor mechanism, without involvement of neurotrophins. Adenosine and adenosine agonists can activate Trk receptor phosphorylation specifically through the seven transmembrane spanning adenosine 2A (A2A) receptor. Several features of Trk receptor transactivation are noteworthy and differ significantly from other transactivation events. Trk receptor transactivation is slower and results in a selective increase in activated Akt. Unlike the biological actions of other tyrosine kinase receptors, increased Trk receptor activity by adenosine resulted in increased cell survival. This article will discuss potential mechanisms by which adenosine can activate trophic responses through Trk tyrosine kinase receptors.