Cross Talk between P2Y₂Nucleotide Receptors and CXC Chemokine Receptor 2 Resulting in Enhanced Ca²⁺Signaling Involves Enhancement of Phospholipase C Activity and Is Enabled by Incremental Ca²⁺Release in Human Embryonic Kidney Cells

Abstract

We have shown previously that activation of endogenously expressed, G␣ q/11 -coupled P2Y 2 nucleotide receptors with UTP reveals an intracellular Ca 2ϩ response to activation of recombinant, G␣ i -coupled CXC chemokine receptor 2 (CXCR2) in human embryonic kidney cells. Here, we characterize further this cross talk and demonstrate that phospholipase C (PLC) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ]-dependent Ca 2ϩ release underlies this potentiation. The putative Ins(1,4,5)P 3 receptor antagonist 2-aminoethoxydiphenyl borane reduced the response to CXCR2 activation by interleukin-8, as did sustained inhibition of phosphatidylinositol 4-kinase with wortmannin, suggesting the involvement of phosphoinositides in the potentiation. Against a Li ϩ block of inositol monophosphatase activity, costimulation of P2Y 2 nucleotide receptors and CXCR2 caused phosphoinositide accumulation that was significantly greater than that after activation of P2Y 2 nucleotide receptors or CXCR2 alone, and was more than additive. Thus, PLC activity, as well as Ca 2ϩ release, was enhanced. In these cells, agonist-mediated Ca 2ϩ release was incremental in nature, suggesting that a potentiation of Ins(1,4,5)P 3 generation in the presence of coactivation of P2Y 2 nucleotide receptors and CXCR2 would be sufficient for additional Ca 2ϩ release. Potentiated Ca 2ϩ signaling by CXCR2 was markedly attenuated by expression of either regulator of G protein signaling 2 or the G␤␥-scavenger G␣ t1 (transducin ␣ subunit), indicating the involvement of G␣ q and G␤␥ subunits, respectively.Through a variety of intracellular signal transduction pathways, diverse ligands of G protein-coupled receptors (GPCRs) are able to regulate many different aspects of cell function. Typically, each GPCR is considered to preferentially activate a specific signal transduction pathway, but it is clear that promiscuity and cross talk can occur. Thus, a GPCR may couple to more than one pathway, whereas activation of a GPCR is often able to influence the signaling by another coexpressed receptor. Such cross talk can have either positive or negative effects on receptor function and may serve to allow coincidence detection, thereby integrating signals from multiple receptor types.A phenomenon of particular interest in terms of positive GPCR cross talk is the enhancement of intracellular Ca 2ϩ release arising as a consequence of the concomitant or sequential stimulation of two types of GPCR that are preferentially coupled to different G proteins. For example, G␣ i/oor G␣ s -coupled receptors can markedly enhance the Ca 2ϩ signaling of simultaneously activated G␣ q/11 -coupled receptors in clonal cell lines (Dickenson and Hill, 1994;Yeo et al., 2001). Such cross talk has also been demonstrated in cells derived from both the central nervous system (Jimenez et al., 1999;Hirono et al., 2001) and peripheral tissues (Shah et al., This work was jointly funded by the Biotechnology and Biological Sciences Research Council and AstraZeneca R&D Charnwood (Loughborough, UK)....

Full and Partial Agonists of Muscarinic M3 Receptors Reveal Single and Oscillatory Ca2+ Responses by β2-Adrenoceptors

“…Ca 2ϩ signaling by ␤ 2 -adrenoceptors was abolished after antagonism of muscarinic receptors with atropine, indicating that concurrent activation of both receptors is required. This is consistent with many examples of cross-talk resulting in enhanced Ca 2ϩ signaling (Dickenson and Hill, 1998;Jiménez et al, 1999;Tanimura et al, 1999;Chan et al, 2000;Short and Taylor, 2000;Yeo et al, 2001;Werry et al, 2003b). …”

“…The generation of [ 3 H]InsP x as an index of phospholipase C activity was determined as described previously (Werry et al, 2003b). In brief, cells were grown Cross-Talk between G␣ q/11 -and G␣ s -Coupled GPCRs for 48 h in the presence of 3 Ci ⅐ ml Ϫ1 myo-[ 3 H]inositol.…”

“…Although some examples of cross-talk are dependent upon enhanced activation of phospholipase C (PLC) and therefore increased generation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P 3 ] to release Ca 2ϩ from the intracellular stores (Dickenson and Hill, 1998;Chan et al, 2000;Werry et al, 2003b), others are independent of enhanced PLC activity, suggesting either increased sensitivity of Ca 2ϩ release channels or alternative release mechanisms (Jiménez et al, 1999;Tanimura et al, 1999;Short and Taylor, 2000;Yeo et al, 2001). Irrespective of the mechanisms involved, the ability of cross-talk to influence intracellular Ca 2ϩ signaling has profound implications for cell function given the diverse cellular events regulated by Ca 2ϩ .…”

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

“…The cross-talk between the P2Y 2 and CXCR2 receptors involved an enhancement of PLC activity (Werry et al, 2003) and that between neuropeptide Y1 and P2Y receptors in CHO-K1 cells was inhibited by ␤␥ subunit scavengers (Selbie et al, 1997). Thus, the most likely possibility is that G protein ␤␥ subunits generated by G i -coupled receptors interact with G protein ␣ subunits generated by the P2Y agonists at the level of PLC␤, thus increasing the production of IP 3 and DAG, with consequent increases in Ca 2ϩ release and PKC activation.…”

Regulator of G-Protein Signaling 4 Suppresses LPS-Induced MUC5AC Overproduction in the Airway

“…RGS proteins were originally discovered as inhibitors of G-protein signaling, since they act as GTPase-activating proteins (GAP). RGS2 has been shown to attenuate P2Y 2 -receptor mediated Ca 21 signaling by Gai-coupled chemokine receptor 2 (CXCR2) in HEK293 cells (13). The role of RGS in LPS-induced, ATPdependent MUC5AC expression has not been explored in human airway epithelial cells.…”