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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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IP3-mediated Ca2+ release from WT and Homer 2−/− cells. Cells from (A) WT and (B) Homer 2−/− mice were permeabilized with SLO and allowed to reduce [Ca2+] of the incubation medium to ∼75 nM. Next, Ca2+ release was measured by adding increasing concentrations of IP3 (A and B, arrows). C summarizes the results obtained in five experiments with cells prepared from three WT and three Homer2−/− mice. The results are expressed as the mean ± SEM. In D–H, cells from (D and E) WT and (F and G) Homer 2−/− were used to record the Ca2+-activated Cl− current using whole cell recording. In E and G, the pipette solution also contained 20 μM 2,4,5 IP3. Where indicated by the bars, the cells were stimulated with (D and F) 0.5 μM or (D–G) 1 mM carbachol. H presents the mean ± SEM of the frequency of IP3-evoked [Ca2+]i oscillations in seven experiments with WT cells obtained from three mice and five experiments with Homer 2−/− cells obtained from two mice.
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fig7: IP3-mediated Ca2+ release from WT and Homer 2−/− cells. Cells from (A) WT and (B) Homer 2−/− mice were permeabilized with SLO and allowed to reduce [Ca2+] of the incubation medium to ∼75 nM. Next, Ca2+ release was measured by adding increasing concentrations of IP3 (A and B, arrows). C summarizes the results obtained in five experiments with cells prepared from three WT and three Homer2−/− mice. The results are expressed as the mean ± SEM. In D–H, cells from (D and E) WT and (F and G) Homer 2−/− were used to record the Ca2+-activated Cl− current using whole cell recording. In E and G, the pipette solution also contained 20 μM 2,4,5 IP3. Where indicated by the bars, the cells were stimulated with (D and F) 0.5 μM or (D–G) 1 mM carbachol. H presents the mean ± SEM of the frequency of IP3-evoked [Ca2+]i oscillations in seven experiments with WT cells obtained from three mice and five experiments with Homer 2−/− cells obtained from two mice.

Mentions: The frequency of Ca2+ oscillations is controlled by a biochemical mechanism through the production of IP3 (Luo et al., 2001) and by several biophysical mechanisms that control the activity of the IP3Rs (Kiselyov et al., 2003). The activity of IP3Rs is regulated by the concentrations of IP3 and [Ca2+]i (Thrower et al., 2001), as well as Ca2+ content in the stores, which increases channel sensitivity to IP3 (Missiaen et al., 1992; Xu et al., 1996b). Because of the increased SERCA2b and Ca2+ content in the stores of Homer2−/− cells, we considered the possibility that Ca2+ release from the stores of these cells was more sensitive to IP3, leading to increased frequency of [Ca2+]i oscillations. The results in Fig. 7 exclude this possibility. Fig. 7 (A–C) shows the same potency for IP3 to release Ca2+ from the stores of streptolysin O (SLO)–permeabilized WT and Homer2−/− cells. In Fig. 7 (D–H), the frequency of IP3-evoked [Ca2+]i oscillations was measured in intact cells. In these experiments, the whole cell mode of the patch clamp technique was used to record the Ca2+-activated Cl− current as a reporter of [Ca2+]i (Zeng et al., 1996). The controls in Fig. 7 (D and F) show that [Ca2+]i oscillations evoked by 0.5 μM carbachol occurred at a higher frequency (1.86- ± 0.26-fold, n = 7) in Homer2−/− compared with control cells, whereas the response to maximal agonist stimulation was the same in both cell types. Long-lasting, IP3-evoked [Ca2+]i oscillations were induced by infusing the cells with 20 μM of the nonhydrolyzable IP3 analogue, 2,4,5 IP3. Fig. 7 (E–G) shows that IP3 evoked [Ca2+]i oscillations with indistinguishable frequency in the two cell types. The implications of the findings in Fig. 7 are that the function of the IP3Rs is the same in cells from WT and Homer2−/− mice and that the increased GPCR sensitivity to agonist stimulation in Homer2−/− cells is due to regulation by Homer 2 of a step upstream of Ca2+ release by IP3.


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)

IP3-mediated Ca2+ release from WT and Homer 2−/− cells. Cells from (A) WT and (B) Homer 2−/− mice were permeabilized with SLO and allowed to reduce [Ca2+] of the incubation medium to ∼75 nM. Next, Ca2+ release was measured by adding increasing concentrations of IP3 (A and B, arrows). C summarizes the results obtained in five experiments with cells prepared from three WT and three Homer2−/− mice. The results are expressed as the mean ± SEM. In D–H, cells from (D and E) WT and (F and G) Homer 2−/− were used to record the Ca2+-activated Cl− current using whole cell recording. In E and G, the pipette solution also contained 20 μM 2,4,5 IP3. Where indicated by the bars, the cells were stimulated with (D and F) 0.5 μM or (D–G) 1 mM carbachol. H presents the mean ± SEM of the frequency of IP3-evoked [Ca2+]i oscillations in seven experiments with WT cells obtained from three mice and five experiments with Homer 2−/− cells obtained from two mice.
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Related In: Results  -  Collection

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fig7: IP3-mediated Ca2+ release from WT and Homer 2−/− cells. Cells from (A) WT and (B) Homer 2−/− mice were permeabilized with SLO and allowed to reduce [Ca2+] of the incubation medium to ∼75 nM. Next, Ca2+ release was measured by adding increasing concentrations of IP3 (A and B, arrows). C summarizes the results obtained in five experiments with cells prepared from three WT and three Homer2−/− mice. The results are expressed as the mean ± SEM. In D–H, cells from (D and E) WT and (F and G) Homer 2−/− were used to record the Ca2+-activated Cl− current using whole cell recording. In E and G, the pipette solution also contained 20 μM 2,4,5 IP3. Where indicated by the bars, the cells were stimulated with (D and F) 0.5 μM or (D–G) 1 mM carbachol. H presents the mean ± SEM of the frequency of IP3-evoked [Ca2+]i oscillations in seven experiments with WT cells obtained from three mice and five experiments with Homer 2−/− cells obtained from two mice.
Mentions: The frequency of Ca2+ oscillations is controlled by a biochemical mechanism through the production of IP3 (Luo et al., 2001) and by several biophysical mechanisms that control the activity of the IP3Rs (Kiselyov et al., 2003). The activity of IP3Rs is regulated by the concentrations of IP3 and [Ca2+]i (Thrower et al., 2001), as well as Ca2+ content in the stores, which increases channel sensitivity to IP3 (Missiaen et al., 1992; Xu et al., 1996b). Because of the increased SERCA2b and Ca2+ content in the stores of Homer2−/− cells, we considered the possibility that Ca2+ release from the stores of these cells was more sensitive to IP3, leading to increased frequency of [Ca2+]i oscillations. The results in Fig. 7 exclude this possibility. Fig. 7 (A–C) shows the same potency for IP3 to release Ca2+ from the stores of streptolysin O (SLO)–permeabilized WT and Homer2−/− cells. In Fig. 7 (D–H), the frequency of IP3-evoked [Ca2+]i oscillations was measured in intact cells. In these experiments, the whole cell mode of the patch clamp technique was used to record the Ca2+-activated Cl− current as a reporter of [Ca2+]i (Zeng et al., 1996). The controls in Fig. 7 (D and F) show that [Ca2+]i oscillations evoked by 0.5 μM carbachol occurred at a higher frequency (1.86- ± 0.26-fold, n = 7) in Homer2−/− compared with control cells, whereas the response to maximal agonist stimulation was the same in both cell types. Long-lasting, IP3-evoked [Ca2+]i oscillations were induced by infusing the cells with 20 μM of the nonhydrolyzable IP3 analogue, 2,4,5 IP3. Fig. 7 (E–G) shows that IP3 evoked [Ca2+]i oscillations with indistinguishable frequency in the two cell types. The implications of the findings in Fig. 7 are that the function of the IP3Rs is the same in cells from WT and Homer2−/− mice and that the increased GPCR sensitivity to agonist stimulation in Homer2−/− cells is due to regulation by Homer 2 of a step upstream of Ca2+ release by IP3.

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