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Homer 2 tunes G protein-coupled receptors stimulus intensity by regulating RGS proteins and PLCbeta GAP activities.

Shin DM, Dehoff M, Luo X, Kang SH, Tu J, Nayak SK, Ross EM, Worley PF, Muallem S - J. Cell Biol. (2003)

Bottom Line: In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations.Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo.Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, Brain Korea 21 Project of Medical Sciences, Yonsei University, Seoul, South Korea.

ABSTRACT
Homers are scaffolding proteins that bind G protein-coupled receptors (GPCRs), inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), ryanodine receptors, and TRP channels. However, their role in Ca2+ signaling in vivo is not known. Characterization of Ca2+ signaling in pancreatic acinar cells from Homer2-/- and Homer3-/- mice showed that Homer 3 has no discernible role in Ca2+ signaling in these cells. In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLCbeta and evoke Ca2+ release and oscillations. This was not due to aberrant localization of IP3Rs in cellular microdomains or IP3R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo. Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLCbeta GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca2+]i oscillations.

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Agonist-evoked [Ca2+]i oscillations in WT and Homer 2−/− cells. As indicated in the figure, cells from WT and Homer 2−/− mice were stimulated with (A) 20 pM CCK, or (C) 50 and 100 pM BS to measure [Ca2+]i oscillations. The frequency of the oscillations, determined in cells prepared from three to five mice as indicated in the figure, are summarized in B for 1 μM carbachol and 20 pM CCK, and in D for the two concentrations of BS, and are given as the mean ± SEM.
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fig6: Agonist-evoked [Ca2+]i oscillations in WT and Homer 2−/− cells. As indicated in the figure, cells from WT and Homer 2−/− mice were stimulated with (A) 20 pM CCK, or (C) 50 and 100 pM BS to measure [Ca2+]i oscillations. The frequency of the oscillations, determined in cells prepared from three to five mice as indicated in the figure, are summarized in B for 1 μM carbachol and 20 pM CCK, and in D for the two concentrations of BS, and are given as the mean ± SEM.

Mentions: To probe Ca2+ signaling further, we examined the response to increasing concentrations of carbachol. At low concentrations, agonists evoke [Ca2+]i oscillations. The frequency, and in some cases, the amplitude, of the oscillations increases with increased agonist concentration until at a high enough agonist concentration the oscillations merge into a single transient increase in [Ca2+]i (Berridge, 1993). This pattern is shown in Fig. 5 (D–F), left traces, for cells from WT mice. At 1 μM, carbachol induced low frequency oscillations (see Fig. 6 B for summary). The residual Ca2+ content in the stores was estimated by discharging it by exposing the cells to 1 mM carbachol and 10 μM CPA. 2.5 μM carbachol caused a substantial initial increase in [Ca2+]i that was followed by high frequency–low amplitude oscillations. Finally, 10 μM carbachol evoked a large increase in [Ca2+]i with high frequency oscillations superimposed on the downward stroke of [Ca2+]i. 10 μM carbachol released ∼70% of stored Ca2+. Remarkably, deletion of Homer 2 increased the response at all carbachol concentrations between 1 and 10 μM. Thus, 1 μM carbachol induced a response in Homer2−/− cells similar to that induced by 2.5 μM carbachol in WT cells. 2.5 μM carbachol caused a transient increase in [Ca2+]i while releasing ∼60% of stored Ca2+, whereas 10 μM carbachol mobilized the entire intracellular Ca2+ pool of Homer2−/− cells, similar to the effect of 100 μM carbachol in WT cells.


Homer 2 tunes G protein-coupled receptors stimulus intensity by regulating RGS proteins and PLCbeta GAP activities.

Shin DM, Dehoff M, Luo X, Kang SH, Tu J, Nayak SK, Ross EM, Worley PF, Muallem S - J. Cell Biol. (2003)

Agonist-evoked [Ca2+]i oscillations in WT and Homer 2−/− cells. As indicated in the figure, cells from WT and Homer 2−/− mice were stimulated with (A) 20 pM CCK, or (C) 50 and 100 pM BS to measure [Ca2+]i oscillations. The frequency of the oscillations, determined in cells prepared from three to five mice as indicated in the figure, are summarized in B for 1 μM carbachol and 20 pM CCK, and in D for the two concentrations of BS, and are given as the mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172804&req=5

fig6: Agonist-evoked [Ca2+]i oscillations in WT and Homer 2−/− cells. As indicated in the figure, cells from WT and Homer 2−/− mice were stimulated with (A) 20 pM CCK, or (C) 50 and 100 pM BS to measure [Ca2+]i oscillations. The frequency of the oscillations, determined in cells prepared from three to five mice as indicated in the figure, are summarized in B for 1 μM carbachol and 20 pM CCK, and in D for the two concentrations of BS, and are given as the mean ± SEM.
Mentions: To probe Ca2+ signaling further, we examined the response to increasing concentrations of carbachol. At low concentrations, agonists evoke [Ca2+]i oscillations. The frequency, and in some cases, the amplitude, of the oscillations increases with increased agonist concentration until at a high enough agonist concentration the oscillations merge into a single transient increase in [Ca2+]i (Berridge, 1993). This pattern is shown in Fig. 5 (D–F), left traces, for cells from WT mice. At 1 μM, carbachol induced low frequency oscillations (see Fig. 6 B for summary). The residual Ca2+ content in the stores was estimated by discharging it by exposing the cells to 1 mM carbachol and 10 μM CPA. 2.5 μM carbachol caused a substantial initial increase in [Ca2+]i that was followed by high frequency–low amplitude oscillations. Finally, 10 μM carbachol evoked a large increase in [Ca2+]i with high frequency oscillations superimposed on the downward stroke of [Ca2+]i. 10 μM carbachol released ∼70% of stored Ca2+. Remarkably, deletion of Homer 2 increased the response at all carbachol concentrations between 1 and 10 μM. Thus, 1 μM carbachol induced a response in Homer2−/− cells similar to that induced by 2.5 μM carbachol in WT cells. 2.5 μM carbachol caused a transient increase in [Ca2+]i while releasing ∼60% of stored Ca2+, whereas 10 μM carbachol mobilized the entire intracellular Ca2+ pool of Homer2−/− cells, similar to the effect of 100 μM carbachol in WT cells.

Bottom Line: In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations.Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo.Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, Brain Korea 21 Project of Medical Sciences, Yonsei University, Seoul, South Korea.

ABSTRACT
Homers are scaffolding proteins that bind G protein-coupled receptors (GPCRs), inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), ryanodine receptors, and TRP channels. However, their role in Ca2+ signaling in vivo is not known. Characterization of Ca2+ signaling in pancreatic acinar cells from Homer2-/- and Homer3-/- mice showed that Homer 3 has no discernible role in Ca2+ signaling in these cells. In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLCbeta and evoke Ca2+ release and oscillations. This was not due to aberrant localization of IP3Rs in cellular microdomains or IP3R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo. Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLCbeta GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca2+]i oscillations.

Show MeSH
Related in: MedlinePlus