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| Specific Observation(s) |
Source of Data |
| LPA (2.5µM) + ISO (50nM) = greater than additive effect |
RAW 2-Ligand Screen: cAMP
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| C5a (250nM) + ISO (50nM) = greater than additive effect |
RAW 2-Ligand Screen: cAMP
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| 2MA (500µM) + ISO (50nM) = greater than additive effect |
RAW 2-Ligand Screen: cAMP
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| UDP (25µM) + ISO (50nM) = greater than additive effect |
RAW 2-Ligand Screen: cAMP
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| PAF (100nM) + ISO (50nM) = greater than additive effect |
RAW 2-Ligand Screen: cAMP
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| RAW 264.7 cell probably express adenylyl cyclases 2,3,7, and 9 |
Affymetrix Microarray Data
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| Abstract |
| Five of the GPCR ligands that synergistically increase cAMP responses to isoproterenol induce activtion of a variety of different trimeric G proteins, including Gi (C5a, LPA, PAF), Gq/11 (LPA, 2MA, UDP, PAF), and G12/13 (LPA). [Note: a separate hypothesis deals with a sixth GPCR ligand, S1P, which also synergistically increases isoproterenol-stimulated cAMP accumulation.] The RAW 264.7 cell’s rich complement of adenylyl cyclases provides several potential mechanisms for synergy. Isoproterenol, acting via the beta2-adrenoceptor, presumably activates all four isoforms via Gs. Cyclase isoforms 2 and 7 are activated by Gbetagamma or by PKC-mediated phosphorylation; the effect of Gbetagamma (but not that of PKC) requires simultaneous stimulation by Gs-alpha); in addition, PKC-dependent phosphorylation inhibits the Gbetagamma-mediated activation (1). Isoform 3 is stimulated by Ca2+-CaM; isoform 9 appears to respond only to Gs-alpha (1). We propose that cyclase isoform 2 mediates synergism with isoproterenol in two ways: (a) C5a activates Gi and synergizes by increasing stimulation by Gbetamma; (b) UDP, LPA, 2MA, and PAF activate Gq/11 to potentiate cAMP elevation via PKC-dependent phosphorylation. Present data do not rule out additional possible contributions from cyclase isoform 3, which could respond to Ca2+/CaM stimulation in response to any of the five ligands. Lin et al. reported (2, 3) that LPA potentiates the cAMP response to isoproterenol in RAW264.7 cells, and suggested that this effect is mediated by atypical PKC isoforms acting on adenylyl cyclase 2. Evidence included: failure of pertussis toxin to block potentiation; documented expression in RAW264.7 cells of both classical and atypical PKCs isoforms (alpha/betaII/delta and epsilon/mu/lambda/zeta, respectively); activation of atypical PKC isoforms by LPA; failure of 24-hour PMA treatment to block LPA/isoproterenol synergy or activation of atypical PKCs; and effects of selective PI3K inhibitors. [We note, however, that these workers applied LPA at concentrations of 30-50 micromolar (vs. 2.5 micromolar in our experiments) and that in their hands 3 micromolar LPA did not significantly elevate atypical PKC activity.] |
| References |
| 1. Sunahara RK and Taussig R (2002) Isoforms of mammalian adenylyl cyclase: multiplicities of signaling. Mol Interv. 2(3), 168-184. |
| 2. Lin WW and Chen BC (1998) Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages. Br J Pharmacol. 125(7), 1601-1609. |
| 3. Lin WW, Chang SH, and Wang SM (1999) Roles of atypical protein kinase C in lysophosphatidic acid-induced type II adenylyl cyclase activation in RAW 264.7 macrophages. Br J Pharmacol 128(6), 1189-1198. |
| Suggested Future Experiments |
| Our hypothesis can be tested by applying perturbations with toxins, drugs and RNAi knockdowns targeted against specific signaling proteins. Trimeric G protein and their subunits. By hypothesis, pertussis toxin treatment should block Gi-mediated synergy of isoproterenol with C5a but not synergy of isoproterenol with the other ligands. Gi-alpha2 or Gbeta2 knockdowns should selectively reduce the C5a effect, while knockdowns of Gq-alpha, G11-alpha, or both together should reduce effects of UDP, 2MA, LPA, and PAF, but not C5a. Cyclase isoforms 2 and 7. Knockdown of either isoform or of the two in combination should reduce the synergy of isoproterenol with all five of the other ligands, although these knockdowns may severely reduce the isoproterenol-stimulated cAMP response, making it difficult to assess synergy. Cyclase isoform 3 and intracellular Ca2+. Failure of an isoform 3 knockdown to block effects of UDP, LPA, PAF, and 2MA would suggest that their synergistic effects are not mediated by intracellular Ca2+. Involvement of this isoform in a synergistic effect would also be less likely if the synergy is not prevented by reducing intracellular Ca2+ accumulation in response to the same four ligands — with thapsigargin ± removal of extracellular Ca2+ or by knockdown of the appropriate phospholipase(s), etc. PKC isoforms. PMA experiments and isoform-selective drugs could help to make a case for or against effects of classical or atypical PKC isoforms in all of the synergies. The same is true of RNAi knockdowns, with the obvious qualification that inferences may be confounded by the RAW264.7 cell’s complement of potentially redundant PKC isoforms. |
| Discussion |
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[03 MAY 2005]
Richard Clark, PhD,
Univ Tx Hlth Ctr Houston Med Sch
Just to support Henry's cautionary note about 50 micromolar LPA: From our comparison of LPA with a range of other related lipids on Gi -mediated inhibition of AC we concluded that huge detergent-like artifacts occur when LPA or other lipids are used at a concentration greater than 5 micromolar.
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