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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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Effect of RGS4 on Ca2+ signaling in WT and Homer 2āˆ’/āˆ’ cells. Cells from (Aā€“C) WT or (Dā€“F) Homer 2āˆ’/āˆ’ mice were infused with (A and D) a control pipette solution, or pipette solutions containing (B and E) 0.25 nM or (C and F) 1 nM RGS4. About 7 min after brake-in to allow equilibration of RGS4 between pipette solution and cytosol, the cells were stimulated with 0.5 Ī¼M or 1 mM carbachol, as indicated by the bars. The number of experiments performed with similar results is indicated in parenthesis next to each trace.
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fig9: Effect of RGS4 on Ca2+ signaling in WT and Homer 2āˆ’/āˆ’ cells. Cells from (Aā€“C) WT or (Dā€“F) Homer 2āˆ’/āˆ’ mice were infused with (A and D) a control pipette solution, or pipette solutions containing (B and E) 0.25 nM or (C and F) 1 nM RGS4. About 7 min after brake-in to allow equilibration of RGS4 between pipette solution and cytosol, the cells were stimulated with 0.5 Ī¼M or 1 mM carbachol, as indicated by the bars. The number of experiments performed with similar results is indicated in parenthesis next to each trace.

Mentions: To test the second possibility, we measured the inhibition of Ca2+ signaling by RGS4, which was infused into the cells using a patch pipette. Due to expression of several RGS proteins in one cell and the lack of information as to the specific RGS protein in each Ca2+-signaling complex, it was not possible to manipulate the native RGS proteins activity. Instead, we measured the ability of exogenous RGS4 to inhibit Ca2+ signaling. Fig. 9 (Aā€“C) shows that in WT cells, 0.25 nM RGS4 abolished [Ca2+]i oscillations in response to 0.5 Ī¼M carbachol and inhibited >90% of the response to maximal stimulation with 1 mM carbachol. In contrast, in Homer2āˆ’/āˆ’ cells, 0.25 nM RGS4 only partially inhibited the response to maximal carbachol stimulation (Fig. 9 E). The response to 1 mM carbachol was partially blocked by up to 1 nM RGS4 (Fig. 9 F), which converted the sustained response to [Ca2+]i oscillations. Complete inhibition of the response to 1 mM carbachol in Homer2āˆ’/āˆ’ cells was observed at RGS4 concentrations above 2.5 nM (n = 4).


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)

Effect of RGS4 on Ca2+ signaling in WT and Homer 2āˆ’/āˆ’ cells. Cells from (Aā€“C) WT or (Dā€“F) Homer 2āˆ’/āˆ’ mice were infused with (A and D) a control pipette solution, or pipette solutions containing (B and E) 0.25 nM or (C and F) 1 nM RGS4. About 7 min after brake-in to allow equilibration of RGS4 between pipette solution and cytosol, the cells were stimulated with 0.5 Ī¼M or 1 mM carbachol, as indicated by the bars. The number of experiments performed with similar results is indicated in parenthesis next to each trace.
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Related In: Results  -  Collection

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fig9: Effect of RGS4 on Ca2+ signaling in WT and Homer 2āˆ’/āˆ’ cells. Cells from (Aā€“C) WT or (Dā€“F) Homer 2āˆ’/āˆ’ mice were infused with (A and D) a control pipette solution, or pipette solutions containing (B and E) 0.25 nM or (C and F) 1 nM RGS4. About 7 min after brake-in to allow equilibration of RGS4 between pipette solution and cytosol, the cells were stimulated with 0.5 Ī¼M or 1 mM carbachol, as indicated by the bars. The number of experiments performed with similar results is indicated in parenthesis next to each trace.
Mentions: To test the second possibility, we measured the inhibition of Ca2+ signaling by RGS4, which was infused into the cells using a patch pipette. Due to expression of several RGS proteins in one cell and the lack of information as to the specific RGS protein in each Ca2+-signaling complex, it was not possible to manipulate the native RGS proteins activity. Instead, we measured the ability of exogenous RGS4 to inhibit Ca2+ signaling. Fig. 9 (Aā€“C) shows that in WT cells, 0.25 nM RGS4 abolished [Ca2+]i oscillations in response to 0.5 Ī¼M carbachol and inhibited >90% of the response to maximal stimulation with 1 mM carbachol. In contrast, in Homer2āˆ’/āˆ’ cells, 0.25 nM RGS4 only partially inhibited the response to maximal carbachol stimulation (Fig. 9 E). The response to 1 mM carbachol was partially blocked by up to 1 nM RGS4 (Fig. 9 F), which converted the sustained response to [Ca2+]i oscillations. Complete inhibition of the response to 1 mM carbachol in Homer2āˆ’/āˆ’ cells was observed at RGS4 concentrations above 2.5 nM (n = 4).

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