Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+

Werry, Tim D.; Wilkinson, Graeme F.; Willars, Gary B.

doi:10.1042/bj20030312

Cited by 166 publications

(135 citation statements)

References 205 publications

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“…Because S1P5 receptors are expressed on oligodendrocytes, and S1P5 receptors are thought to play a role in oligodendrocyte differentiation and survival, we focused on the development of S1P5 agonists. By using a highthroughput screening calcium mobilization assay with GPCR priming and FLIPR technology, [12] we discovered benzamide 1, which has good in vitro potency toward the S1P5 receptor (EC 50 = 270 nm), but has modest selectivity against S1P1 (EC 50 = 3140 nm) and S1P4 (EC 50 = 100 nm). Herein we report our studies of various benzamide modifications carried out to improve the selectivity, bioactivity, pharmacokinetic properties, and ancillary profile of 1, ultimately resulting in the discovery of potent and very selective S1P5 agonists.…”

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confidence: 99%

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

et al. 2010

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The immunomodulatory drug fingolimod (FTY720, 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol), derived from a fungal metabolite myriocin), is phosphorylated in vivo by sphingosine kinases to produce (R)-FTY720-phosphate (FTY720-P). [1,2] FTY720-P activates sphingosine-1-phosphate (S1P) receptors S1P1, S1P3, S1P4, and S1P5 at low nanomolar concentrations and is inactive toward the S1P2 receptor.[1] The FTY720-P-mediated activation of the S1P1 receptor on lymphocytes induces receptor internalization, which attenuates T-cell response to S1P gradients, preventing their egress from secondary lymphoid tissues. [3] In addition to playing a role in the immune system, all S1P receptors except S1P4 are also found differentially expressed in the central nervous system [4] and on various tumor cell types. [5,6] Although the precise regulation of these receptors by locally released S1P remains unclear, S1P receptors are thought to play a role in such events as astrocyte migration, [7] oligodendrocyte differentiation, and cell survival [8,9] and neurogenesis. [10,11] To assess the relevance of individual S1P receptor subtypes for the activity of FTY720-P, selective agonists are required. Because S1P5 receptors are expressed on oligodendrocytes, and S1P5 receptors are thought to play a role in oligodendrocyte differentiation and survival, we focused on the development of S1P5 agonists. By using a highthroughput screening calcium mobilization assay with GPCR priming and FLIPR technology, [12] we discovered benzamide 1, which has good in vitro potency toward the S1P5 receptor (EC 50 = 270 nm), but has modest selectivity against S1P1 (EC 50 = 3140 nm) and S1P4 (EC 50 = 100 nm). Herein we report our studies of various benzamide modifications carried out to improve the selectivity, bioactivity, pharmacokinetic properties, and ancillary profile of 1, ultimately resulting in the discovery of potent and very selective S1P5 agonists.To guide the optimization process, homology models of all S1P receptors were built from a crystal structure of bovine rhodopsin (PDB ID: 1F88). [13] Docking experiments of 1 into these models revealed a possible location of the binding site, some essential features of the interactions, and indicated potential regions for gaining selectivity and improving potency. In these complexes (Figure 1), 1 adopts a twisted conformation with the aniline ring,~708 out of the benzamide plane and stabilized by a hydrogen bond between the aniline NH group and the amide carbonyl. In the S1P5 receptor complex, the amide group forms a hydrogen bond with OG1-Thr120. The benzamide phenyl ring lies in a large hydrophobic pocket surrounded by Phe196, Phe201, Phe268, Leu119, Trp264, Leu267, and Leu271. The aniline ring undergoes a T-shaped interaction with Phe116 and hydrophobic contacts with Leu271 and Leu292. The ortho-methyl substituents fill a small pocket formed by Tyr89, Val115, and Leu292 on one side, and sit at the face of Phe196 on the other side. Inspection of sequence alignments (Figure 2) revealed two positions, o...

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Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

et al. 2010

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“…To solve this problem, we used the antagonizing peptide, WRW4, which does not activate but does bind FPRL1 (16). Desensitization events between two GPCRs usually occur via agonist-induced receptor activation (20). In addition, we show that the FPRL1-specific antagonist peptide, WRW4, can inhibit PACAP27-induced calcium signaling.…”

Section: Discussionmentioning

confidence: 99%

“…Cross-desensitization provides a straightforward and powerful means of illustrating receptor sharing. However, some GPCR groups do codesensitize via single receptor activation for reasons, like receptor oligomerization, sequestration, and others (20). To solve this problem, we used the antagonizing peptide, WRW4, which does not activate but does bind FPRL1 (16).…”

Section: Discussionmentioning

confidence: 99%

Pituitary Adenylate Cyclase-Activating Polypeptide 27 Is a Functional Ligand for Formyl Peptide Receptor-Like 1

Kim

Lee

Kim

et al. 2006

The Journal of Immunology

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Although the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in the regulation of several immune responses, its target receptors and signaling mechanisms have yet to be fully elucidated in immune cells. In this study, we found that PACAP27, but not PACAP38, specifically stimulated intracellular calcium mobilization and ERK phosphorylation in human neutrophils. Moreover, formyl peptide receptor-like 1 (FPRL1) was identified as a PACAP27 receptor, and PACAP27 was found to selectively stimulate intracellular calcium increase in FPRL1-transfected rat basophile leukocytes-2H3 cell lines. In addition, PACAP27-induced calcium increase and ERK phosphorylation were specifically inhibited by an FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp-Trp (WRW4), thus supporting the notion that PACAP27 acts on FPRL1. In terms of the functional role of PACAP27, we found that the peptide stimulated CD11b surface up-regulation and neutrophil chemotactic migration, and that these responses were completely inhibited by WRW4. The interaction between PACAP27 and FPRL1 was analyzed further using truncated PACAPs and chimeric PACAPs using vasoactive intestinal peptide, and the C-terminal region of PACAP27 was found to perform a vital function in the activation of FPRL1. Taken together, our study suggests that PACAP27 activates phagocytes via FPRL1 activation, and that this results in proinflammatory behavior, involving chemotaxis and the up-regulation of CD11b.

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“…Synergy required simultaneous receptor activation, and it affected both the initial release of Ca 2ϩ from intracellular stores and the sustained elevation in Ca 2ϩ levels. Similar to synergy in other cell types, synergy in macrophages occurred at the level of inositol 1,4,5-trisphosphate production (10,(12)(13)(14)(15)(16). Although macrophages express multiple PLC␤ isoforms, these were used selectively in Ca 2ϩ signaling and synergy.…”

Section: Cross-talk Between G␣ I -And G␣ Q -Linked G-protein-coupledmentioning

confidence: 99%

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

Rebres

Roach

Fraser

et al. 2011

Journal of Biological Chemistry

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Cross-talk between G␣ i -and G␣ q -linked G-protein-coupled receptors yields synergistic Ca2؉ responses in a variety of cell types. Prior studies have shown that synergistic Ca 2؉ responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase C␤3 (PLC␤3), with a possible contribution of PLC␤2, whereas signaling through PLC␤4 interferes with synergy. We here show that synergy can be induced by the combination of G␤␥ and G␣ q activation of a single PLC␤ isoform. Synergy was absent in macrophages lacking both PLC␤2 and PLC␤3, but it was fully reconstituted following transduction with PLC␤3 alone. Mechanisms of PLC␤-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLC␤2. RNAi-mediated knockdown of endogenous PLC␤s demonstrated that synergy in these cells was dependent on PLC␤3, but PLC␤1 and PLC␤4 did not contribute, and overexpression of either isoform inhibited Ca 2؉ synergy. When synergy was blocked by RNAi of endogenous PLC␤3, it could be reconstituted by expression of either human PLC␤3 or mouse PLC␤2. In contrast, it could not be reconstituted by human PLC␤3 with a mutation of the Y box, which disrupted activation by G␤␥, and it was only partially restored by human PLC␤3 with a mutation of the C terminus, which partly disrupted activation by G␣ q . Thus, both G␤␥ and G␣ q contribute to activation of PLC␤3 in cells for Ca 2؉ synergy. We conclude that Ca 2؉ synergy between G␣ i -coupled and G␣ q -coupled receptors requires the direct action of both G␤␥ and G␣ q on PLC␤ and is mediated primarily by PLC␤3, although PLC␤2 is also competent.Phosphoinositide-specific phospholipase C (PLC) 5 -dependent Ca 2ϩ responses are stimulated by several types of cell surface receptors, including members of the GPCR family. G␣ q -linked GPCRs stimulate Ca 2ϩ responses in most cells, whereas G␣ i -linked receptor stimulation of Ca 2ϩ release is variably seen, and the factors that determine this variation are not well understood (1-3). Simultaneous activation of G␣ iand G␣ q -linked GPCRs has revealed pathway cross-talk, resulting in synergistic Ca 2ϩ responses in a variety of primary and cultured cell types (4 -10). Proposed mechanisms have included effects on receptor availability, changes in G-protein turnover, increased availability of the PLC␤ substrate phosphatidylinositol 4,5-diphosphate, increased sensitivity of intracellular stores to inositol 1,4,5-trisphosphate, and enhanced linkage of intracellular Ca 2ϩ release to Ca 2ϩ influx (5). From these disparate results, it is clear that the mechanisms for Ca 2ϩ synergy may vary between systems and contexts. We recently demonstrated that G␣ i -and G␣ q -coupled GPCRs synergize in stimulating a rise in [Ca 2ϩ ] i in macrophages (11). Synergy required simultaneous receptor activation, and it affected both the initial release of Ca 2ϩ from intracellular stores and the sustained elevation in Ca 2ϩ levels. Similar to synergy in other cell types, synergy in macrophages occurred at the level of inositol 1,4,5-tri...

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Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+

Cited by 166 publications

References 205 publications

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

Pituitary Adenylate Cyclase-Activating Polypeptide 27 Is a Functional Ligand for Formyl Peptide Receptor-Like 1

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

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