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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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Ca2+ signaling in cells from Homer 3−/− mice. A and B show immunolocalization of Homer 3 in WT and Homer 3−/− cells, respectively. Examples of the Ca2+ response of WT (bold lines) or Homer 3−/− cells (dashed lines) to stimulation with (C) 1 mM carbachol or (D) 50 pM CCK is shown. The traces in D are with acini from two separate mice. Similar results were obtained using various protocols with cells prepared from nine WT and nine Homer 3−/− mice.
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fig4: Ca2+ signaling in cells from Homer 3−/− mice. A and B show immunolocalization of Homer 3 in WT and Homer 3−/− cells, respectively. Examples of the Ca2+ response of WT (bold lines) or Homer 3−/− cells (dashed lines) to stimulation with (C) 1 mM carbachol or (D) 50 pM CCK is shown. The traces in D are with acini from two separate mice. Similar results were obtained using various protocols with cells prepared from nine WT and nine Homer 3−/− mice.

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)

Ca2+ signaling in cells from Homer 3−/− mice. A and B show immunolocalization of Homer 3 in WT and Homer 3−/− cells, respectively. Examples of the Ca2+ response of WT (bold lines) or Homer 3−/− cells (dashed lines) to stimulation with (C) 1 mM carbachol or (D) 50 pM CCK is shown. The traces in D are with acini from two separate mice. Similar results were obtained using various protocols with cells prepared from nine WT and nine Homer 3−/− mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172804&req=5

fig4: Ca2+ signaling in cells from Homer 3−/− mice. A and B show immunolocalization of Homer 3 in WT and Homer 3−/− cells, respectively. Examples of the Ca2+ response of WT (bold lines) or Homer 3−/− cells (dashed lines) to stimulation with (C) 1 mM carbachol or (D) 50 pM CCK is shown. The traces in D are with acini from two separate mice. Similar results were obtained using various protocols with cells prepared from nine WT and nine Homer 3−/− mice.
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