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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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Characterization of Ca2+ signaling in Homer 2−/− cells. (A and B) Cells from WT (solid lines) and Homer 2−/− (dashed lines) mice were used to measure the [Ca2+]i response to stimulation with 1 mM carbachol in the presence (black bars) or absence (open bars) of (A) 2 mM medium Ca2+. Where indicated by the bold lines, the cells were also treated with 10 μM CPA to inhibit the SERCA pumps. C shows the mean ± SEM of the indicated number of experiments performed with cells from different mice. D–F compare the responses obtained with WT (left) and Homer 2−/− (right) cells stimulated with (D) 1, (E) 2.5, or (F) 10 μM carbachol. The residual Ca2+ pool after each stimulation was estimated by completely discharging it with 1 mM carbachol and 100 μM CPA. Each panel shows two traces from two cells present in the same recording field. Similar results were obtained with cells prepared from three different mice of each phenotype.
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fig5: Characterization of Ca2+ signaling in Homer 2−/− cells. (A and B) Cells from WT (solid lines) and Homer 2−/− (dashed lines) mice were used to measure the [Ca2+]i response to stimulation with 1 mM carbachol in the presence (black bars) or absence (open bars) of (A) 2 mM medium Ca2+. Where indicated by the bold lines, the cells were also treated with 10 μM CPA to inhibit the SERCA pumps. C shows the mean ± SEM of the indicated number of experiments performed with cells from different mice. D–F compare the responses obtained with WT (left) and Homer 2−/− (right) cells stimulated with (D) 1, (E) 2.5, or (F) 10 μM carbachol. The residual Ca2+ pool after each stimulation was estimated by completely discharging it with 1 mM carbachol and 100 μM CPA. Each panel shows two traces from two cells present in the same recording field. Similar results were obtained with cells prepared from three different mice of each phenotype.

Mentions: Expression of Homer 3 at the basal pole of pancreatic acinar cells, its absence from the apical pole, and the specificity of Homer 3 localization is further illustrated in Fig. 4 (A and B). A comparison of Ca2+ signals obtained in WT and Homer 3−/− cells stimulated with high agonist concentration to trigger a single [Ca2+]i transient (Fig. 4 C) or stimulated with low agonist concentration to evoke [Ca2+]i oscillations (Fig. 4 D) revealed no clear difference in any parameter of the Ca2+ signals. Likewise, no differences were observed in the Ca2+ signals recorded from WT and Homer 3−/− cells using the protocols in Figs. 4 and 5. Deletion of Homer 3 also had no effect on localization of Ca2+-signaling proteins in the apical pole of pancreatic, submandibular, and parotid gland acinar and duct cells, as was found for Homer 2−/− (unpublished data). Hence, it appears that Homer 3 does not participate directly in controlling GPCR-dependent Ca2+ signaling in the cells and tissue types examined. This is not surprising considering the differential localization of Ca2+-signaling complexes and Homer 3. The role of Homer 3 in cell function remains to be defined. Preliminary experiments with cells from Homer1−/− mice showed that deletion of Homer 1 did not affect agonist-stimulated IP3 production or Ca2+ mobilization in pancreatic acini (unpublished data), a phenotype different from that of cells from Homer 2−/− mice.


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)

Characterization of Ca2+ signaling in Homer 2−/− cells. (A and B) Cells from WT (solid lines) and Homer 2−/− (dashed lines) mice were used to measure the [Ca2+]i response to stimulation with 1 mM carbachol in the presence (black bars) or absence (open bars) of (A) 2 mM medium Ca2+. Where indicated by the bold lines, the cells were also treated with 10 μM CPA to inhibit the SERCA pumps. C shows the mean ± SEM of the indicated number of experiments performed with cells from different mice. D–F compare the responses obtained with WT (left) and Homer 2−/− (right) cells stimulated with (D) 1, (E) 2.5, or (F) 10 μM carbachol. The residual Ca2+ pool after each stimulation was estimated by completely discharging it with 1 mM carbachol and 100 μM CPA. Each panel shows two traces from two cells present in the same recording field. Similar results were obtained with cells prepared from three different mice of each phenotype.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Characterization of Ca2+ signaling in Homer 2−/− cells. (A and B) Cells from WT (solid lines) and Homer 2−/− (dashed lines) mice were used to measure the [Ca2+]i response to stimulation with 1 mM carbachol in the presence (black bars) or absence (open bars) of (A) 2 mM medium Ca2+. Where indicated by the bold lines, the cells were also treated with 10 μM CPA to inhibit the SERCA pumps. C shows the mean ± SEM of the indicated number of experiments performed with cells from different mice. D–F compare the responses obtained with WT (left) and Homer 2−/− (right) cells stimulated with (D) 1, (E) 2.5, or (F) 10 μM carbachol. The residual Ca2+ pool after each stimulation was estimated by completely discharging it with 1 mM carbachol and 100 μM CPA. Each panel shows two traces from two cells present in the same recording field. Similar results were obtained with cells prepared from three different mice of each phenotype.
Mentions: Expression of Homer 3 at the basal pole of pancreatic acinar cells, its absence from the apical pole, and the specificity of Homer 3 localization is further illustrated in Fig. 4 (A and B). A comparison of Ca2+ signals obtained in WT and Homer 3−/− cells stimulated with high agonist concentration to trigger a single [Ca2+]i transient (Fig. 4 C) or stimulated with low agonist concentration to evoke [Ca2+]i oscillations (Fig. 4 D) revealed no clear difference in any parameter of the Ca2+ signals. Likewise, no differences were observed in the Ca2+ signals recorded from WT and Homer 3−/− cells using the protocols in Figs. 4 and 5. Deletion of Homer 3 also had no effect on localization of Ca2+-signaling proteins in the apical pole of pancreatic, submandibular, and parotid gland acinar and duct cells, as was found for Homer 2−/− (unpublished data). Hence, it appears that Homer 3 does not participate directly in controlling GPCR-dependent Ca2+ signaling in the cells and tissue types examined. This is not surprising considering the differential localization of Ca2+-signaling complexes and Homer 3. The role of Homer 3 in cell function remains to be defined. Preliminary experiments with cells from Homer1−/− mice showed that deletion of Homer 1 did not affect agonist-stimulated IP3 production or Ca2+ mobilization in pancreatic acini (unpublished data), a phenotype different from that of cells from Homer 2−/− mice.

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