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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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Expression of Ca2+ transporters in WT and Homer 2−/− cells. (A) The level of SERCA2b was analyzed in extracts prepared from pancreatic acini of four WT and four Homer 2−/− mice. Extracts prepared from the brain of five WT and five Homer 2−/− mice were also used to analyze the levels of (B) SERCA2b, (C) IP3R1, (D) IP3R3, and (E) PMCA. Expression was analyzed by densitometry and was unaltered for all Ca2+ transporters, except (F) SERCA2b. The results in F are expressed as the mean ± SEM.
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fig3: Expression of Ca2+ transporters in WT and Homer 2−/− cells. (A) The level of SERCA2b was analyzed in extracts prepared from pancreatic acini of four WT and four Homer 2−/− mice. Extracts prepared from the brain of five WT and five Homer 2−/− mice were also used to analyze the levels of (B) SERCA2b, (C) IP3R1, (D) IP3R3, and (E) PMCA. Expression was analyzed by densitometry and was unaltered for all Ca2+ transporters, except (F) SERCA2b. The results in F are expressed as the mean ± SEM.

Mentions: Next, we examined whether deletion of Homer 2 affected expression of the Ca2+ transporters that generate the Ca2+ signal (Fig. 3). Although all proteins could be detected, attempts to quantify protein expression by Western blot using membranes prepared from pancreatic acini were largely unsuccessful due to excessive protein degradation. As reported before, this was particularly the case for all IP3Rs (Lee et al., 1997b). Reliable results could be obtained only for SERCA2b, the expression level of which increased 2.2-fold in pancreatic acinar cells from Homer 2-deficient mice (Fig. 3 A). Similar results were obtained with parotid gland (not depicted) and brain membranes (Fig. 3 B). The immunolocalization in Fig. 2 suggests that deletion of Homer 2 has minimal or no effect on expression of all IP3R isoforms. This was further tested in brain extracts. For the most part, the levels of IP3Rs were unaffected, although a slight increase in IP3R1 may have occurred in Homer2−/− cells (Fig. 3, C and D). Similarly, expression of PMCA was unaltered in Homer2−/− cells (Fig. 3 E). These findings imply that the expression and retention of IP3Rs in cellular microdomains do not require the expression of Homer 2. This conclusion can probably be extended to other components of the Ca2+-signaling complex.


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)

Expression of Ca2+ transporters in WT and Homer 2−/− cells. (A) The level of SERCA2b was analyzed in extracts prepared from pancreatic acini of four WT and four Homer 2−/− mice. Extracts prepared from the brain of five WT and five Homer 2−/− mice were also used to analyze the levels of (B) SERCA2b, (C) IP3R1, (D) IP3R3, and (E) PMCA. Expression was analyzed by densitometry and was unaltered for all Ca2+ transporters, except (F) SERCA2b. The results in F are expressed as the mean ± SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172804&req=5

fig3: Expression of Ca2+ transporters in WT and Homer 2−/− cells. (A) The level of SERCA2b was analyzed in extracts prepared from pancreatic acini of four WT and four Homer 2−/− mice. Extracts prepared from the brain of five WT and five Homer 2−/− mice were also used to analyze the levels of (B) SERCA2b, (C) IP3R1, (D) IP3R3, and (E) PMCA. Expression was analyzed by densitometry and was unaltered for all Ca2+ transporters, except (F) SERCA2b. The results in F are expressed as the mean ± SEM.
Mentions: Next, we examined whether deletion of Homer 2 affected expression of the Ca2+ transporters that generate the Ca2+ signal (Fig. 3). Although all proteins could be detected, attempts to quantify protein expression by Western blot using membranes prepared from pancreatic acini were largely unsuccessful due to excessive protein degradation. As reported before, this was particularly the case for all IP3Rs (Lee et al., 1997b). Reliable results could be obtained only for SERCA2b, the expression level of which increased 2.2-fold in pancreatic acinar cells from Homer 2-deficient mice (Fig. 3 A). Similar results were obtained with parotid gland (not depicted) and brain membranes (Fig. 3 B). The immunolocalization in Fig. 2 suggests that deletion of Homer 2 has minimal or no effect on expression of all IP3R isoforms. This was further tested in brain extracts. For the most part, the levels of IP3Rs were unaffected, although a slight increase in IP3R1 may have occurred in Homer2−/− cells (Fig. 3, C and D). Similarly, expression of PMCA was unaltered in Homer2−/− cells (Fig. 3 E). These findings imply that the expression and retention of IP3Rs in cellular microdomains do not require the expression of Homer 2. This conclusion can probably be extended to other components of the Ca2+-signaling complex.

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