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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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Interaction of Homers with PLCβ and effect of Homer 2 on RGS4 and PLCβ GAP activity and PLCβ PIP2 hydrolytic activity in a phospholipid reconstitution system. (A and B) Soluble lysates from (A) pancreatic acini or (B) adult rat forebrain were assayed for binding to GST-Homer fusion proteins. (A) Homer 2, but not Homer 1c and the mutant Homer 1aW24A, binds PLCβ. (B, top) PLCβ binds full-length Homer 2 preferentially to WT Homer 1 EVH domain or to the Homer 1 mutants W24A and G89N. Input lanes are 10% of that used for the binding assay. (Middle) mGluR5 binds strongly to both Homer 2 and WT Homer 1, but not to the mutants. (Bottom) Coomassie stain of gel showing GST proteins. (C and D) Recombinant, purified M1 receptor, and Gαβγ heterotrimer were reconstituted into liposomes containing PIP2 and used for measurements of (C) GTPase activity or (D) PIP2 hydrolysis. GTPase activity was measured in the absence and presence of 2–5 nM RGS4 or 2 nM PLCβ, and in the absence and presence of 250 nM Homer 2 or 1. GTPase activity was initiated by stimulation with 1 mM carbachol. C shows the results of six experiments with RGS4, four experiments with PLCβ performed in duplicate with two separate batches of Homer 2, and two experiments performed in duplicate with Homer 1 and RGS4 or PLCβ. The proteoliposomes were also used to assay PLCβ activity by measuring hydrolysis of (D) PIP2. The results in D are from seven experiments performed in duplicates and with two batches of Homer 2. Stimulation by Homer 2 was calculated as a percentage of the activity measured in the absence of Homers 1 and 2 and the presence of RGS4 or PLCβ. GAP activity in the absence of Homer 2 and the presence of RGS4 or PLCβ averaged 4.62 ± 0.7 (n = 9) and 4.16 ± 0.34 (n = 7) pmol Pi released/min/pmol Gq, respectively. Basal PLC PIP2 hydrolytic activity averaged 2.01 ± 0.12 (n = 10) pmol IP3 released/min/pmol PLCβ. The results are expressed as the mean ± SEM.
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fig10: Interaction of Homers with PLCβ and effect of Homer 2 on RGS4 and PLCβ GAP activity and PLCβ PIP2 hydrolytic activity in a phospholipid reconstitution system. (A and B) Soluble lysates from (A) pancreatic acini or (B) adult rat forebrain were assayed for binding to GST-Homer fusion proteins. (A) Homer 2, but not Homer 1c and the mutant Homer 1aW24A, binds PLCβ. (B, top) PLCβ binds full-length Homer 2 preferentially to WT Homer 1 EVH domain or to the Homer 1 mutants W24A and G89N. Input lanes are 10% of that used for the binding assay. (Middle) mGluR5 binds strongly to both Homer 2 and WT Homer 1, but not to the mutants. (Bottom) Coomassie stain of gel showing GST proteins. (C and D) Recombinant, purified M1 receptor, and Gαβγ heterotrimer were reconstituted into liposomes containing PIP2 and used for measurements of (C) GTPase activity or (D) PIP2 hydrolysis. GTPase activity was measured in the absence and presence of 2–5 nM RGS4 or 2 nM PLCβ, and in the absence and presence of 250 nM Homer 2 or 1. GTPase activity was initiated by stimulation with 1 mM carbachol. C shows the results of six experiments with RGS4, four experiments with PLCβ performed in duplicate with two separate batches of Homer 2, and two experiments performed in duplicate with Homer 1 and RGS4 or PLCβ. The proteoliposomes were also used to assay PLCβ activity by measuring hydrolysis of (D) PIP2. The results in D are from seven experiments performed in duplicates and with two batches of Homer 2. Stimulation by Homer 2 was calculated as a percentage of the activity measured in the absence of Homers 1 and 2 and the presence of RGS4 or PLCβ. GAP activity in the absence of Homer 2 and the presence of RGS4 or PLCβ averaged 4.62 ± 0.7 (n = 9) and 4.16 ± 0.34 (n = 7) pmol Pi released/min/pmol Gq, respectively. Basal PLC PIP2 hydrolytic activity averaged 2.01 ± 0.12 (n = 10) pmol IP3 released/min/pmol PLCβ. The results are expressed as the mean ± SEM.

Mentions: To corroborate this unexpected consequence of Homer 2 deletion, we measured the effect of recombinant Homer 2 on the GAP activity of RGS4 in an in vitro reconstitution system. The reconstitution system included the M1 muscarinic receptor and the heterotrimer Gαqβγ reconstituted into liposomes (Biddlecome et al., 1996). The Gαq GTPase activity in this system is dependent on receptor stimulation by carbachol and is inhibited by the muscarinic receptor antagonist atropine (Zeng et al., 1998). Fig. 10 C shows that Homer 2 increased RGS4 GAP activity by ∼90%. Importantly, stimulation of RGS4 GAP activity was specific to Homer 2, as Homer 1 had no effect.


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)

Interaction of Homers with PLCβ and effect of Homer 2 on RGS4 and PLCβ GAP activity and PLCβ PIP2 hydrolytic activity in a phospholipid reconstitution system. (A and B) Soluble lysates from (A) pancreatic acini or (B) adult rat forebrain were assayed for binding to GST-Homer fusion proteins. (A) Homer 2, but not Homer 1c and the mutant Homer 1aW24A, binds PLCβ. (B, top) PLCβ binds full-length Homer 2 preferentially to WT Homer 1 EVH domain or to the Homer 1 mutants W24A and G89N. Input lanes are 10% of that used for the binding assay. (Middle) mGluR5 binds strongly to both Homer 2 and WT Homer 1, but not to the mutants. (Bottom) Coomassie stain of gel showing GST proteins. (C and D) Recombinant, purified M1 receptor, and Gαβγ heterotrimer were reconstituted into liposomes containing PIP2 and used for measurements of (C) GTPase activity or (D) PIP2 hydrolysis. GTPase activity was measured in the absence and presence of 2–5 nM RGS4 or 2 nM PLCβ, and in the absence and presence of 250 nM Homer 2 or 1. GTPase activity was initiated by stimulation with 1 mM carbachol. C shows the results of six experiments with RGS4, four experiments with PLCβ performed in duplicate with two separate batches of Homer 2, and two experiments performed in duplicate with Homer 1 and RGS4 or PLCβ. The proteoliposomes were also used to assay PLCβ activity by measuring hydrolysis of (D) PIP2. The results in D are from seven experiments performed in duplicates and with two batches of Homer 2. Stimulation by Homer 2 was calculated as a percentage of the activity measured in the absence of Homers 1 and 2 and the presence of RGS4 or PLCβ. GAP activity in the absence of Homer 2 and the presence of RGS4 or PLCβ averaged 4.62 ± 0.7 (n = 9) and 4.16 ± 0.34 (n = 7) pmol Pi released/min/pmol Gq, respectively. Basal PLC PIP2 hydrolytic activity averaged 2.01 ± 0.12 (n = 10) pmol IP3 released/min/pmol PLCβ. The results are expressed as the mean ± SEM.
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fig10: Interaction of Homers with PLCβ and effect of Homer 2 on RGS4 and PLCβ GAP activity and PLCβ PIP2 hydrolytic activity in a phospholipid reconstitution system. (A and B) Soluble lysates from (A) pancreatic acini or (B) adult rat forebrain were assayed for binding to GST-Homer fusion proteins. (A) Homer 2, but not Homer 1c and the mutant Homer 1aW24A, binds PLCβ. (B, top) PLCβ binds full-length Homer 2 preferentially to WT Homer 1 EVH domain or to the Homer 1 mutants W24A and G89N. Input lanes are 10% of that used for the binding assay. (Middle) mGluR5 binds strongly to both Homer 2 and WT Homer 1, but not to the mutants. (Bottom) Coomassie stain of gel showing GST proteins. (C and D) Recombinant, purified M1 receptor, and Gαβγ heterotrimer were reconstituted into liposomes containing PIP2 and used for measurements of (C) GTPase activity or (D) PIP2 hydrolysis. GTPase activity was measured in the absence and presence of 2–5 nM RGS4 or 2 nM PLCβ, and in the absence and presence of 250 nM Homer 2 or 1. GTPase activity was initiated by stimulation with 1 mM carbachol. C shows the results of six experiments with RGS4, four experiments with PLCβ performed in duplicate with two separate batches of Homer 2, and two experiments performed in duplicate with Homer 1 and RGS4 or PLCβ. The proteoliposomes were also used to assay PLCβ activity by measuring hydrolysis of (D) PIP2. The results in D are from seven experiments performed in duplicates and with two batches of Homer 2. Stimulation by Homer 2 was calculated as a percentage of the activity measured in the absence of Homers 1 and 2 and the presence of RGS4 or PLCβ. GAP activity in the absence of Homer 2 and the presence of RGS4 or PLCβ averaged 4.62 ± 0.7 (n = 9) and 4.16 ± 0.34 (n = 7) pmol Pi released/min/pmol Gq, respectively. Basal PLC PIP2 hydrolytic activity averaged 2.01 ± 0.12 (n = 10) pmol IP3 released/min/pmol PLCβ. The results are expressed as the mean ± SEM.
Mentions: To corroborate this unexpected consequence of Homer 2 deletion, we measured the effect of recombinant Homer 2 on the GAP activity of RGS4 in an in vitro reconstitution system. The reconstitution system included the M1 muscarinic receptor and the heterotrimer Gαqβγ reconstituted into liposomes (Biddlecome et al., 1996). The Gαq GTPase activity in this system is dependent on receptor stimulation by carbachol and is inhibited by the muscarinic receptor antagonist atropine (Zeng et al., 1998). Fig. 10 C shows that Homer 2 increased RGS4 GAP activity by ∼90%. Importantly, stimulation of RGS4 GAP activity was specific to Homer 2, as Homer 1 had no effect.

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