Limits...
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.

Show MeSH
Localization of Homer proteins and IP3Rs in WT and Homer 2−/− cells. Acini from WT and Homer 2−/− mice were stained for (A and B) Homer 1, (C and D) Homer 2, (E and F) Homer 3, (G and H) IP3R, (I and J) IP3R2, and (K and L) IP3R3. Note lack of effect of Homer 2 deletion on expression and localization of the other Homers and, in particular, IP3Rs.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172804&req=5

fig2: Localization of Homer proteins and IP3Rs in WT and Homer 2−/− cells. Acini from WT and Homer 2−/− mice were stained for (A and B) Homer 1, (C and D) Homer 2, (E and F) Homer 3, (G and H) IP3R, (I and J) IP3R2, and (K and L) IP3R3. Note lack of effect of Homer 2 deletion on expression and localization of the other Homers and, in particular, IP3Rs.

Mentions: The most basic function of a scaffolding protein is targeting and retention of its binding partners in a defined microdomain. Therefore, we first compared localization and expression of the Homers and IP3Rs in cells from wild-type (WT) and mutant mice. Fig. 1 describes the generation of the Homer 2−/− mice and shows that the gene was deleted (B), and the mRNA (B) and protein (C) could not be detected. Fig. 2 illustrates the localization of the three Homer isoforms and of IP3Rs in cells from WT and Homer2−/− mice. It is evident that Homers 1 and 2 are expressed exclusively in the apical pole of pancreatic acinar cells (Fig. 2, A–D), the site enriched in all three isoforms of IP3Rs (Fig. 2, G–L) and other Ca2+-signaling proteins (Lee et al., 1997a,b; Shin et al., 2001; Zhao et al., 2001) and from which Ca2+ waves emanate (Xu et al., 1996a; Shin et al., 2001). On the other hand, Homer 3 is expressed in the basal pole (Fig. 2, E and F). Deletion of Homer 2 did not affect expression of Homer 1 or 3. Most notably, deletion of Homer 2 had no effect on the localization of any IP3R isoform. This was probably not due to the compensatory effect of other Homers because preliminary experiments with pancreatic acini from one mouse from which Homers 1, 2, and 3 were deleted showed no obvious effect of Homers deletion on IP3Rs localization. This was unexpected in view of the binding of IP3Rs to the EVH domain of Homers (Tu et al., 1998).


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)

Localization of Homer proteins and IP3Rs in WT and Homer 2−/− cells. Acini from WT and Homer 2−/− mice were stained for (A and B) Homer 1, (C and D) Homer 2, (E and F) Homer 3, (G and H) IP3R, (I and J) IP3R2, and (K and L) IP3R3. Note lack of effect of Homer 2 deletion on expression and localization of the other Homers and, in particular, IP3Rs.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Localization of Homer proteins and IP3Rs in WT and Homer 2−/− cells. Acini from WT and Homer 2−/− mice were stained for (A and B) Homer 1, (C and D) Homer 2, (E and F) Homer 3, (G and H) IP3R, (I and J) IP3R2, and (K and L) IP3R3. Note lack of effect of Homer 2 deletion on expression and localization of the other Homers and, in particular, IP3Rs.
Mentions: The most basic function of a scaffolding protein is targeting and retention of its binding partners in a defined microdomain. Therefore, we first compared localization and expression of the Homers and IP3Rs in cells from wild-type (WT) and mutant mice. Fig. 1 describes the generation of the Homer 2−/− mice and shows that the gene was deleted (B), and the mRNA (B) and protein (C) could not be detected. Fig. 2 illustrates the localization of the three Homer isoforms and of IP3Rs in cells from WT and Homer2−/− mice. It is evident that Homers 1 and 2 are expressed exclusively in the apical pole of pancreatic acinar cells (Fig. 2, A–D), the site enriched in all three isoforms of IP3Rs (Fig. 2, G–L) and other Ca2+-signaling proteins (Lee et al., 1997a,b; Shin et al., 2001; Zhao et al., 2001) and from which Ca2+ waves emanate (Xu et al., 1996a; Shin et al., 2001). On the other hand, Homer 3 is expressed in the basal pole (Fig. 2, E and F). Deletion of Homer 2 did not affect expression of Homer 1 or 3. Most notably, deletion of Homer 2 had no effect on the localization of any IP3R isoform. This was probably not due to the compensatory effect of other Homers because preliminary experiments with pancreatic acini from one mouse from which Homers 1, 2, and 3 were deleted showed no obvious effect of Homers deletion on IP3Rs localization. This was unexpected in view of the binding of IP3Rs to the EVH domain of Homers (Tu et al., 1998).

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