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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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PLCβ activity in agonist and GTPγS stimulated cells from WT and Homer 2−/− cells. (A) Intact cells from WT (squares) or Homer 2−/− (circles) mice were stimulated with various [carbachol] for 5 s, the reactions were stopped, and IP3 was extracted and measured as described in Materials and methods. The figure shows the mean ± SEM of four experiments performed in duplicates with four different cell preparations. (B) Cells from three WT (open symbols) and three Homer 2−/− mice (closed symbols) were permeabilized with SLO in intracellular-like medium and stimulated with the indicated [GTPγS] for 15 s at 37°C. This procedure requires a large amount of cells to ensure reduction of medium [Ca2+] to the nanometer range (Fig. 7) and, therefore, single determinations were performed at each [GTPγS]. Different symbols represent experiments with different cell preparations. The same open and closed symbols indicate experiments performed in parallel with cells from WT and Homer 2−/− mice.
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fig8: PLCβ activity in agonist and GTPγS stimulated cells from WT and Homer 2−/− cells. (A) Intact cells from WT (squares) or Homer 2−/− (circles) mice were stimulated with various [carbachol] for 5 s, the reactions were stopped, and IP3 was extracted and measured as described in Materials and methods. The figure shows the mean ± SEM of four experiments performed in duplicates with four different cell preparations. (B) Cells from three WT (open symbols) and three Homer 2−/− mice (closed symbols) were permeabilized with SLO in intracellular-like medium and stimulated with the indicated [GTPγS] for 15 s at 37°C. This procedure requires a large amount of cells to ensure reduction of medium [Ca2+] to the nanometer range (Fig. 7) and, therefore, single determinations were performed at each [GTPγS]. Different symbols represent experiments with different cell preparations. The same open and closed symbols indicate experiments performed in parallel with cells from WT and Homer 2−/− mice.

Mentions: Enhanced activity of the biochemical component of Ca2+ signaling in Homer2−/− cells implies enhanced activation of PLCβ and IP3 production, which can account for the increased signal intensity and frequency of Ca2+ oscillations. This was tested by measuring the dose response for carbachol-stimulated IP3 production. Notably, Fig. 8 A shows that deletion of Homer 2 increased the potency of carbachol in stimulating IP3 production about fivefold without affecting maximally stimulated IP3 production. The same phenomenon was observed using two submaximal and one maximal concentration of CCK and BS (unpublished data).


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)

PLCβ activity in agonist and GTPγS stimulated cells from WT and Homer 2−/− cells. (A) Intact cells from WT (squares) or Homer 2−/− (circles) mice were stimulated with various [carbachol] for 5 s, the reactions were stopped, and IP3 was extracted and measured as described in Materials and methods. The figure shows the mean ± SEM of four experiments performed in duplicates with four different cell preparations. (B) Cells from three WT (open symbols) and three Homer 2−/− mice (closed symbols) were permeabilized with SLO in intracellular-like medium and stimulated with the indicated [GTPγS] for 15 s at 37°C. This procedure requires a large amount of cells to ensure reduction of medium [Ca2+] to the nanometer range (Fig. 7) and, therefore, single determinations were performed at each [GTPγS]. Different symbols represent experiments with different cell preparations. The same open and closed symbols indicate experiments performed in parallel with cells from WT and Homer 2−/− mice.
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Related In: Results  -  Collection

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fig8: PLCβ activity in agonist and GTPγS stimulated cells from WT and Homer 2−/− cells. (A) Intact cells from WT (squares) or Homer 2−/− (circles) mice were stimulated with various [carbachol] for 5 s, the reactions were stopped, and IP3 was extracted and measured as described in Materials and methods. The figure shows the mean ± SEM of four experiments performed in duplicates with four different cell preparations. (B) Cells from three WT (open symbols) and three Homer 2−/− mice (closed symbols) were permeabilized with SLO in intracellular-like medium and stimulated with the indicated [GTPγS] for 15 s at 37°C. This procedure requires a large amount of cells to ensure reduction of medium [Ca2+] to the nanometer range (Fig. 7) and, therefore, single determinations were performed at each [GTPγS]. Different symbols represent experiments with different cell preparations. The same open and closed symbols indicate experiments performed in parallel with cells from WT and Homer 2−/− mice.
Mentions: Enhanced activity of the biochemical component of Ca2+ signaling in Homer2−/− cells implies enhanced activation of PLCβ and IP3 production, which can account for the increased signal intensity and frequency of Ca2+ oscillations. This was tested by measuring the dose response for carbachol-stimulated IP3 production. Notably, Fig. 8 A shows that deletion of Homer 2 increased the potency of carbachol in stimulating IP3 production about fivefold without affecting maximally stimulated IP3 production. The same phenomenon was observed using two submaximal and one maximal concentration of CCK and BS (unpublished data).

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