Article |
Address correspondence to Shmuel Muallem, Dept. of Physiology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9040. Tel.: (214) 648-2593. Fax: (214) 648-8879. E-mail: Shmuel.Muallem{at}UTSouthwestern.edu; or Paul F. Worley, 600 North Wolfe St., Baltimore, MD 21205. Tel.: (410) 502-5489. Fax: (410) 614-8423. E-mail: pworley{at}jhmi.edu
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Abstract |
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Key Words: Homers; GPCR; Ca2+ signaling; regulation; IP3
* Abbreviations used in this paper: BS, bombesin; CPA, cyclopiazonic acid; GAP, GTPase-activating protein; GPCR, G proteincoupled receptors; IP3, inositol 1,4,5-triphosphate; IP3R, IP3 receptor; pAb, polyclonal antibody; PIP2, phosphatidylinositol-bisphosphate; PMCA, plasma membrane Ca2+ ATPase; RGS, regulators of G proteins signaling; SERCA, sarco/endoplasmic reticulum Ca2+ ATPase; SLO, streptolysin O; WT, wild-type.
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Introduction |
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The highly coordinated [Ca2+]i oscillations and waves require polarized expression of Ca2+-signaling proteins, their organization into complexes, and regulation of each component within the signaling complex. Indeed, Ca2+-signaling proteins are clustered in microdomains of polarized cells, such as the pre- and postsynaptic membranes in neurons (Hering and Sheng, 2001) and the apical pole of secretory cells (Kiselyov et al., 2003). Signaling complexes are assembled with the aid of scaffolding proteins that express multiple proteinprotein interacting domains (Hering and Sheng, 2001; Minke and Cook, 2002). The role of scaffolding proteins in tyrosine kinase receptors (Hunter, 2000) and cAMP/PKA-mediated signaling (Smith and Scott, 2002) is well characterized. Much less is known about scaffolding proteins in Ca2+ signaling. In synapses, PSD-95, SHANK, GRIP, and probably other scaffolds, participate in assembly of signaling complexes, including Ca2+ signaling (Hering and Sheng, 2001). InaD is the scaffold that assembles Ca2+-signaling complexes in Drosophila photoreceptors (Minke and Cook, 2002). However, the primary scaffolding protein that assembles Ca2+-signaling complexes in nonneuronal cells is not known. Homer proteins have recently emerged as attractive candidates (Fagni et al., 2002). Homers are scaffolding proteins that are composed of an EVH proteinbinding domain, a coiled-coil multimerization domain, and a leucine zipper (Fagni et al., 2002). The EVH domain binds the GPCR mGluR1/5, IP3Rs, ryanodine receptors, and probably other proteins involved in Ca2+ signaling (Tu et al., 1998; Xiao et al., 1998, 2000). However, the present work reveals that Homers may not function as simple scaffolds, as deletion of Homer 2 or 3 did not disrupt polarized localization of IP3Rs and other Ca2+-signaling proteins in pancreatic acini, but rather affected the efficiency of signal transduction.
G proteins amplify and transduce signals from the receptor to the appropriate effector, and are, thus, a central regulatory site of signal transduction efficiency. Activation of G proteins involves a receptor-catalyzed GDP-GTP exchange reaction on the subunit to release G
·GTP and Gß
(Gilman, 1987), which, in turn, activate separate effector proteins (Gudermann et al., 1997). The off reaction entails the hydrolysis of GTP and reassembly of the G
·GDPß
heterotrimer. This reaction is accelerated by two separate GTPase-activating proteins (GAPs), the PLCß effector protein (Ross, 1995) and the regulators of G proteins signaling (RGS) proteins (Ross and Wilkie, 2000). In vitro (Ross and Wilkie, 2000) and in vivo studies (Cook et al., 2000) suggest that both catalytic mechanisms participate in Ca2+ signaling. Furthermore, regulation of G
q by RGS proteins confers receptor-specific Ca2+ signaling (Xu et al., 1999), drives [Ca2+]i oscillations (Luo et al., 2001), and probably accounts for the oscillation in [IP3] during [Ca2+]i oscillations (Hirose et al., 1999; Nash et al., 2001). [Ca2+]i oscillations due to [IP3] oscillations require cyclical activation and inactivation of RGS and/or PLCß GAP activity. To date, little is known about the regulation of RGS proteins and PLCß GAP activity.
The results reported here show that Homer 3 does not have a major role in Ca2+ signaling in pancreatic acinar cells, whereas Homer 2 regulates GAP activity of both RGS proteins and PLCß. Deletion of Homer 2 resulted in increased potency of agonist-stimulated Ca2+ signaling and, thus, increased frequency of agonist-evoked [Ca2+]i oscillations. This phenotype was traced to reduced GAP activity of exogenous RGS proteins to inhibit Ca2+ signaling in cells from Homer2-/- mice. In support of this mechanism, Homer 2 preferentially binds to PLCß and activates RGS4 and PLCß GAP activity in an in vitro reconstitution system. Thus, Homer 2 tunes the intensity of Ca2+ signaling to regulate [Ca2+]i oscillation frequency and, thus, cell functions regulated by this signaling pathway.
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Results |
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Deletion of Homers does not affect polarized expression of IP3Rs
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, AD), the site enriched in all three isoforms of IP3Rs (Fig. 2, GL) 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).
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To probe Ca2+ signaling further, we examined the response to increasing concentrations of carbachol. At low concentrations, agonists evoke [Ca2+]i oscillations. The frequency, and in some cases, the amplitude, of the oscillations increases with increased agonist concentration until at a high enough agonist concentration the oscillations merge into a single transient increase in [Ca2+]i (Berridge, 1993). This pattern is shown in Fig. 5 (DF), left traces, for cells from WT mice. At 1 µM, carbachol induced low frequency oscillations (see Fig. 6 B for summary). The residual Ca2+ content in the stores was estimated by discharging it by exposing the cells to 1 mM carbachol and 10 µM CPA. 2.5 µM carbachol caused a substantial initial increase in [Ca2+]i that was followed by high frequencylow amplitude oscillations. Finally, 10 µM carbachol evoked a large increase in [Ca2+]i with high frequency oscillations superimposed on the downward stroke of [Ca2+]i. 10 µM carbachol released 70% of stored Ca2+. Remarkably, deletion of Homer 2 increased the response at all carbachol concentrations between 1 and 10 µM. Thus, 1 µM carbachol induced a response in Homer2-/- cells similar to that induced by 2.5 µM carbachol in WT cells. 2.5 µM carbachol caused a transient increase in [Ca2+]i while releasing
60% of stored Ca2+, whereas 10 µM carbachol mobilized the entire intracellular Ca2+ pool of Homer2-/- cells, similar to the effect of 100 µM carbachol in WT cells.
Deletion of Homer 2 increased the potency of all Gq-coupled receptors expressed in acinar cells examined. Fig. 6 shows part of the results obtained with bombesin (BS) and CCK stimulation, focusing on the physiological response of [Ca2+]i oscillations. CCK at 20 pM induced typical baseline [Ca2+]i oscillations in cells from WT mice. The [Ca2+]i oscillations evoked by the same concentration of CCK in cells from Homer2-/- mice occurred at a frequency about twice as higher than those recorded in cells from WT mice (Fig. 6, A and B). The traces in Fig. 6 C show that increasing BS concentration from 50 to 100 pM increased the frequency of the oscillations by 1.7-fold in WT cells. The same fold increase in frequency (1.8) was observed in Homer2-/- cells, but at each BS concentration, the frequency of the oscillations was 2.3-fold higher in Homer2-/- cells. In aggregate, the results of Figs. 5 and 6 show that deletion of Homer 2 increases the potency of agonists to stimulate Ca2+ signaling by GPCRs. The increase in potency is due to a change in a regulatory step common to signaling by GPCRs because the response evoked by all GPCRs examined was affected. In the next stage, we searched for this Homer 2regulated general mechanism.
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To test the second possibility, we measured the inhibition of Ca2+ signaling by RGS4, which was infused into the cells using a patch pipette. Due to expression of several RGS proteins in one cell and the lack of information as to the specific RGS protein in each Ca2+-signaling complex, it was not possible to manipulate the native RGS proteins activity. Instead, we measured the ability of exogenous RGS4 to inhibit Ca2+ signaling. Fig. 9 (AC) shows that in WT cells, 0.25 nM RGS4 abolished [Ca2+]i oscillations in response to 0.5 µM carbachol and inhibited >90% of the response to maximal stimulation with 1 mM carbachol. In contrast, in Homer2-/- cells, 0.25 nM RGS4 only partially inhibited the response to maximal carbachol stimulation (Fig. 9 E). The response to 1 mM carbachol was partially blocked by up to 1 nM RGS4 (Fig. 9 F), which converted the sustained response to [Ca2+]i oscillations. Complete inhibition of the response to 1 mM carbachol in Homer2-/- cells was observed at RGS4 concentrations above 2.5 nM (n = 4).
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The preferential binding of PLCß to signaling complexes containing Homer 2 prompted us to test the effect of Homer 2 on PLCß-GAP activity in the in vitro reconstitution system. Fig. 10 (C and D) shows the effect of Homer 2 on both PLCß GAP activity and PIP2 hydrolysis. Homer 2 stimulated PLCß GAP activity by 140%. This was better than Homer 2 stimulation of RGS4 GAP activity. Like RGS4 stimulation, PLCß stimulation was specific for Homer 2; Homer 1 had no effect on PLCß GAP activity. Homer 2 stimulated PLCß PIP2 hydrolytic activity by only 35%. Homer 2 regulation of RGS4 and PLCß GAP explains at least in part the effect of Homer 2 deletion on Ca2+ signaling.
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Discussion |
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The most notable finding in the present work is that Homer 2 functions to tune the intensity of agonist stimulation through regulation of RGS proteins and PLCß GAP activities. Thus, deletion of Homer 2 left-shift the dose response for agonist-stimulated IP3 production and Ca2+ signaling without affecting the activation of PLCß by activated G, while reducing the effectiveness of exogenous RGS proteins to inhibit Ca2+ signaling in vivo. These findings were corroborated by demonstrating direct activation of RGS proteins and PLCß GAP activities in reconstitution system with recombinant proteins. The physiological significance of these findings is demonstrated by the nearly twofold increased frequency of [Ca2+]i oscillations in Homer2-/- cells.
In a previous work, we showed that RGS proteins provide a biochemical control of [Ca2+]i oscillations (Luo et al., 2001), thus, providing a molecular mechanism for oscillatory changes in [IP3] (Hirose et al., 1999; Nash et al., 2001) to drive [Ca2+]i oscillations. This mechanism explicitly implies that the activity of RGS proteins oscillates to induce oscillations in [IP3] and, consequently, [Ca2+]i oscillations (Luo et al., 2001). How the activity of RGS proteins is regulated during [Ca2+]i oscillations remains unclear. Here, we show that Homer 2 can regulate this activity in vivo and in vitro. Furthermore, we show that PLCß GAP activity is not fixed, but is also regulated by Homer 2. Based on these findings, we propose that Homer 2 participates in regulating the GTPase reaction in the G protein turnover cycle to tune stimulus intensity.
At present, it is not clear how Homer 2 regulates RGS proteins and PLCß GAP activities. However, the findings that Homer 2 preferentially binds PLCß in pancreatic and brain extracts, that stimulation of GAP activity by Homer 2 can be reproduced in the minimal in vitro system, that Homer 2 activates GAP activity of two very different proteins whose only common feature is that they can bind to Gq, and that Homer 2 has proteinprotein binding domains all suggest that Homer 2 may control the proximity of the GAPs to G
q and, thus, the efficiency of the GTPase reaction. This possibility and the molecular details of how Homer 2 controls GAP activity in vivo remain to be determined.
Regulation of RGS proteins and PLCß GAP activities by Homer 2 raises the question of the significance of Homer binding to GPCRs and IP3Rs in Ca2+ signaling. Binding of Homer 2 or any of the Homers to one protein does not exclude binding and regulation of other proteins within the Ca2+-signaling complex. A more intriguing possibility is that different Homer isoforms mediate each of the interactions. In either case, it is clear that although Homer proteins do not appear to be the central scaffolding proteins that assemble the Ca2+-signaling complexes, they play an essential role in controlling Ca2+ signaling. One of their central roles is tuning Ca2+-signaling intensity. By tuning signal intensity, Homer 2 determines the frequency of [Ca2+]i oscillations and, in this way, controls the many cellular functions regulated by [Ca2+]i oscillations (Carafoli, 2002).
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Materials and methods |
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Generation of Homer 2 and 3 mutant mice
The homer 2 targeting construct was generated by inserting transprimer-1 into exon 3 of a 10.2-kb BAC fragment of the mouse homer 2 gene (Fig. 1). PGK-Neo was cloned into the unique PmeI site, and the resulting vector was subcloned into an MC1-TK vector. The resulting targeting construct was linearized and electroporated into R1 ES cells. Cells were selected with G-418 and gancyclovir. Clones were picked, screened by PCR, and confirmed by Southern blotting for homologous recombination. Clones were injected into blastocysts, and chimeras were mated to C57BL/6 mice to produce Homer 2 heterozygotes that were crossed to generate WT and Homer2-/- mice. Homer3-/- mice were generated using a similar strategy as that used to delete Homer2, except that the PGK-Neo cassette was directly inserted into exon 3 of Homer3 at the SmaI site.
Preparation of pancreatic acini
Acini were prepared from the pancreas of WT, Homer2-/-, and Homer3-/- mice by limited collagenase digestion as described previously (Shin et al., 2001). After isolation, the acini were resuspended in a standard solution A ([mM] 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes, pH 7.4 with NaOH, 10 glucose, and 0.1% BSA), and kept on ice until use. Doublet or triplet acinar cell clusters were obtained by incubating a minced pancreas in a 0.025% trypsin, 0.02% EDTA solution for 5 min at 37°C. After washing with solution A supplemented with 0.02% soybean trypsin inhibitor (PSA), doublets and triplets were liberated by a 7-min incubation at 37°C in the same solution that also contained 160 U/ml pure collagenase. The cells were washed with solution A and kept on ice until use.
Measurement of [Ca2+]i
Cells in PSA were incubated with 5 µM Fura2/AM for 30 min at room temperature and washed once with PSA. Samples of cells were plated on glass coverslips that formed the bottom of a perfusion chamber. After 23 min of incubation, to allow cell attachment to the coverslip, the cells were continuously perfused with prewarmed (37°C) solution A at a rate of 5 ml/min (30 vol chamber/min). Agonists were delivered to the cells by inclusion in the perfusate. Fura2 fluorescence was measured at excitation wavelengths of 340 and 380 nm using a PTI image acquisition and analysis system as detailed previously (Shin et al., 2001).
Electrophysiology
The whole cell configuration of the patch clamp technique was used for measurement of Ca2+-activated Cl- current as a reporter of [Ca2+]i next to the PMCA. The experiments were performed with single acinar cells perfused with solution A. The standard pipette solution contained (mM): 140 KCl, 1 MgCl2, 0.1 EGTA, 5 ATP, 10 Hepes (pH 7.3 with KOH) with or without 2,4,5 IP3 or between 0.25 and 10 nM RGS4, as described previously (Zeng et al., 1996). The RGS4 was dialyzed against an ATP-free pipette solution and concentrated to 5 µM with a centricone system. Seals of 610 G
were produced on the cell membrane, and the whole cell configuration was obtained by gentle suction or voltage pulses of 0.5 V for 0.31 ms. The patch clamp output (Axopatch-1B; Axon Instruments, Inc.) was filtered at 20 Hz. Recording was performed with patch clamp 6.0 and a Digi-Data interface (model 1200; Axon Instruments, Inc.). The current was recorded at a holding potential of -40 mV. The oscillation frequency was determined from a stretch of at least 5 min starting at the first full Ca2+ spike. The number of spikes over this time period was counted to determine the number of spikes/minute and results from at least five cells from two mice were used to obtain the results listed in the text and shown in Fig. 7.
Measurement of Ca2+ uptake and release from internal stores
IP3-mediated Ca2+ release from internal stores was measured in SLO-permeabilized cells as described previously (Xu et al., 1996b). In brief, cells washed with a high K+, Chelex-treated medium were added to the same medium containing an ATP regeneration system (comprised of 3 mM ATP, 5 mM MgCl2, 10 mM creatine phosphate, and 5 U/ml creatine kinase), a cocktail of mitochondrial inhibitors, 2 µM Fluo3 and 3 mg/ml SLO (Difco). In this medium, the cells were almost instantaneously permeabilized so that Ca2+ uptake into the ER could be measured immediately. Uptake of Ca2+ into the ER was allowed to continue until medium [Ca2+] was stabilized. Then IP3 was added in increasing concentrations to measure the extent of Ca2+ release and the potency of IP3 in mobilizing Ca2+ from the ER.
Measurement of IP3 mass
IP3 levels were measured by a radioligand assay (Xu et al., 1996b). Intact acini were stimulated with the indicated concentration of agonist for 5 s at 37°C. Permeabilized acini were stimulated with 2.5100 µM GTPS for 15 s at 37°C. Preliminary experiments showed that 1,4,5 IP3 reached maximal level at these incubation times. The reactions were stopped by the addition of 100 µl ice-cold 15% PCA to 100 µl of samples, vigorous mixing, and incubation on ice for at least 10 min to allow precipitation of proteins. Standards of IP3 were prepared in the same manner. The perchloric acid was removed, and IP3 was extracted with 0.2 ml Freon and 0.2 ml tri-n-octylamine. IP3 content in the aqueous phase was measured by displacement of [3H]IP3 using microsomes prepared from bovine brain cerebella.
Western blot
Microsomes were prepared by homogenizing pancreatic acini or brains from WT, Homer2-/-, and Homer3-/- mice in a buffer containing (mM): 100 KCl , 20 Tris-base, pH 7.6 with KOH, 1 EDTA, 1 benzamidine, and 1 PMSF. The homogenates were centrifuged at 1,000 g for 10 min at 4°C. The supernatants were collected and centrifuged at 40,000 g for 30 min. The pellets were resuspended in homogenization buffer and the microsomes were stored at -80°C until use. Microsomes were extracted by a 1-h incubation on ice in a buffer containing (mM): 50 Tris, pH 6.8 with HCl, 150 NaCl, 2 EDTA, 2 EGTA, and 1% Triton X-100 supplemented with protease inhibitors (0.2 mM PMSF, 10 µg/ml leupeptin, 15 µg/ml aprotinin, and 1 mM benzamidine). The extracts were used to separate the proteins by SDS-PAGE and the proteins were probed with a 1:500 dilution of SERCA2b pAb; 1:500 dilution of IP3R1 pAb; and 1:1,000 dilution of IP3R3 and PMCA mAbs.
Pull-down assay
Rat pancreatic acini were used to prepare extracts as described above. Adult rat forebrain was sonicated in four volumes of ice cold PBS with 1% Triton X-100. Soluble extracts were mixed with GST-Homer proteins lined to glutathione beads (Sigma-Aldrich) for 2 h (pancreas) or overnight (brain) at 4°C. Beads were pelleted and washed four times in lysis buffer and eluted with loading buffer followed by SDS-PAGE. The blots were probed for PLCß, mGlu5, or stained with Coomassie blue as before (Tu et al., 1998; Xiao et al., 2000).
Immunocytochemistry
Cells from WT, Homer2-/-, and Homer3-/- mice attached to glass coverslips were fixed and permeabilized with 0.5 ml of cold methanol for 10 min at -20°C, except for the experiments in Figs. 2 and 4 for localization of Homer 3, in which the cells were fixed with 4% formaldehyde for 20 min at room temperature, followed by permeabilization with 0.05% Triton X-100. After removal of methanol or Triton X-100, the cells were washed with PBS and incubated in 0.5 ml PBS containing 50 mM glycine for 10 min at room temperature. This buffer was aspirated and the nonspecific sites were blocked by a 1-h incubation at room temperature with 0.25 ml PBS containing 5% goat serum, 1% BSA, and 0.1% gelatin (blocking medium). The medium was aspirated and replaced with 50 µl of blocking medium containing control serum or a 1:50 dilution of pAbs against Homer 1, 2, or 3, or a 1:100 dilution of pAb against IP3R1, 2, or 3. After incubation with the primary antibodies overnight at 4°C and three washes with the incubation buffer (same as blocking buffer, but without serum), the antibodies were detected with goat antirabbit or antimouse IgG tagged with fluorescein or rhodamine. Images were collected with a confocal microscope (model MRC 1024; Bio-Rad Laboratories).
Measurement of GTPase and PLC activities in vitro
Agonist-stimulated steady-state GTP hydrolysis was measured in reconstituted vesicles that contained recombinant, purified heterotrimeric Gq and M1 acetylcholine receptors at 30°C in the presence and absence of 25 nM RGS4 or PLCß1 (Biddlecome et al., 1996). Homers 1 and 2 (250 nM) were preincubated with the vesicles in the presence of 1 mM carbachol and one of the GAPs for 45 min at 0°C followed by further incubation for 2 min at 30°C (Mukhopadhyay and Ross, 1999). The GTPase reaction was initiated by adding 3 µM -[32P]GTP and was continued for 10 min at 30°C. Reactions were quenched with a cold slurry of Norit in H3PO4 and [32P]Pi was measured in the supernatant.
The effect of Homer 2 on steady-state phospholipase activity of PLCß1 was measured with the same reconstituted phospholipid vesicles, which also contained [3H]PIP2. Homer 2 was preincubated with PLCß1 for 45 min at 0°C. PIP2 hydrolysis was initiated by adding a mixture of the preincubated Homer 2 (250 nM final) and PLCß1 (2 nM final) to the reconstituted vesicles. [3H]IP3 release was measured at 30°C in the presence of 1 mM carbachol, 10 µM GTP, and 10 nM-free Ca2+ as described previously (Biddlecome et al., 1996).
Statistics
When appropriate, results are given as the mean ± SEM of the indicated number of experiments. Statistical significance was evaluated by a two-way ANOVA. All immunostaining experiments were repeated at least five times with similar results.
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Acknowledgments |
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This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute on Deafness and Other Communication Disorders, the National Institute of General Medical Sciences, the National Institute of Drug Abuse, and the National Institute of Mental Health. Support was also provided by a Korea Research Foundation Grant (KRF-2002-015-EP0115) to D.M. Shin.
Submitted: 21 October 2002
Revised: 16 May 2003
Accepted: 23 May 2003
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References |
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---|
Berridge, M.J. 1993. Inositol trisphosphate and calcium signaling. Nature. 361:315325.[CrossRef][Medline]
Biddlecome, G.H., G. Berstein, and E.M. Ross. 1996. Regulation of PLCß1 by Gq and m1 muscarinic cholinergic receptor. Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation. J. Biol. Chem. 271:79998007.
Brini, M., D. Bano, S. Manni, R. Rizzuto, and E. Carafoli. 2000. Effects of PMCA and SERCA pump overexpression on the kinetics of cell Ca2+ signalling. EMBO J. 19:49264935.
Carafoli, E. 2002. Calcium signaling: a tale for all seasons. Proc. Natl. Acad. Sci. USA. 99:11151122.
Cook, B., M. Bar-Yaacov, H. Cohen Ben-Ami, R.E. Goldstein, Z. Paroush, Z. Selinger, and B. Minke. 2000. Phospholipase C and termination of G-protein-mediated signaling in vivo. Nat. Cell Biol. 2:296301.[CrossRef][Medline]
Fagni, L., P.F. Worley, and F. Ango. 2002. Homer as both a scaffold and transduction molecule. Sci. STKE. RE8.
Gilman, A.G. 1987. G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56:615649.[CrossRef][Medline]
Gudermann, T., T. Schoneberg, and G. Schultz. 1997. Functional and structural complexity of signal transduction via G-protein-coupled receptors. Annu. Rev. Neurosci. 20:399427.[CrossRef][Medline]
Hirose, K., S. Kadowaki, M. Tanabe, H. Takeshima, and M. Iino. 1999. Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns. Science. 284:15271530.
Hering, H., and M. Sheng. 2001. Dendritic spines: structure, dynamics and regulation. Nat. Rev. Neurosci. 2:880888.[CrossRef][Medline]
Hunter, T. 2000. Signaling2000 and beyond. Cell. 100: 113127.[Medline]
Kasai, H., Y.X. Li, and Y. Miyashita. 1993. Subcellular distribution of Ca2+ release channels underlying Ca2+ waves and oscillations in exocrine pancreas. Cell. 74:669677.[Medline]
Kiselyov, K., D.M. Shin, and S. Muallem. 2003. Signalling specificity in GPCR-dependent Ca2+ signalling. Cell. Signal. 15:243253.[CrossRef][Medline]
Lee, M.G., X. Xu, W. Zeng, J. Diaz, T. H. Kuo, F. Wuytack, L. Racymaekers, and S. Muallem. 1997a. Polarized expression of Ca2+ pumps in pancreatic and salivary gland cells. Role in initiation and propagation of [Ca2+]i waves. J. Biol. Chem. 272:1577115776.
Lee, M.G., X. Xu, W. Zeng, J. Diaz, R.J. Wojcikiewicz, T.H. Kuo, F. Wuytack, L. Racymaekers, and S. Muallem. 1997b. Polarized expression of Ca2+ channels in pancreatic and salivary gland cells. Correlation with initiation and propagation of [Ca2+]i waves. J. Biol. Chem. 272:1576515770.
Liu, B.F., X. Xu, F. Fridman, S. Muallem, and T.H. Kuo. 1996. Consequences of functional expression of the plasma membrane Ca2+ pump isoform 1a. J. Biol. Chem. 271:55365544.
Luo, X., S. Popov, A.K. Bera, T.M. Wilkie, and S. Muallem. 2001. RGS proteins provide biochemical control of agonist-evoked [Ca2+]i oscillations. Mol. Cell. 7:651660.[Medline]
Minke, B., and B. Cook. 2002. TRP channel proteins and signal transduction. Physiol. Rev. 82:429472.
Missiaen, L., H. De Smedt, G. Droogmans, and R. Casteels. 1992. Ca2+ release induced by inositol 1,4,5-trisphosphate is a steady-state phenomenon controlled by luminal Ca2+ in permeabilized cells. Nature. 357:599602.[CrossRef][Medline]
Mukhopadhyay, S., and E.M. Ross. 1999. Rapid GTP binding and hydrolysis by Gq promoted by receptor and GTPase-activating proteins. Proc. Natl. Acad. Sci. USA. 96:95399544.
Nash, M.S., K.W. Young, R.A. Challiss, and S.R. Nahorski. 2001. Intracellular signaling. Receptor-specific messenger oscillations. Nature. 413:381382.[CrossRef][Medline]
Petersen, O.H., C.C. Petersen, and H. Kasai. 1994. Calcium and hormone action. Annu. Rev. Physiol. 56:297319.[CrossRef][Medline]
Ross, E.M. 1995. G protein GTPase-activating proteins: regulation of speed, amplitude, and signaling selectivity. Recent Prog. Horm. Res. 50:207221.[Medline]
Ross, E.M., and T.M. Wilkie. 2000. GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Biochem. 69:795827.[CrossRef][Medline]
Shin, D.M., X. Luo, T.M. Wilkie, L.J. Miller, A.B. Peck, M.G. Humphreys-Beher, and S. Muallem. 2001. Polarized expression of G protein-coupled receptors and an all-or-none discharge of Ca2+ pools at initiation sites of [Ca2+]i waves in polarized exocrine cells. J. Biol. Chem. 276:4414644156.
Smith, F.D., and J.D. Scott. 2002. Signaling complexes: junctions on the intracellular information super highway. Curr. Biol. 12:R32R40.[CrossRef][Medline]
Thrower, E.C., R.E. Hagar, and B.E. Ehrlich. 2001. Regulation of Ins(1,4,5)P3 receptor isoforms by endogenous modulators. Trends Pharmacol. Sci. 22:580586.[CrossRef][Medline]
Tu, J.C., B. Xiao, J.P. Yuan, A.A. Lanahan, K. Leoffert, M. Li, D.J. Linden, and P.F. Worley. 1998. Homer binds a novel proline-rich motif and links group 1 metabotropic glutamate receptors with IP3 receptors. Neuron. 21:717726.[Medline]
Xiao, B., J.C. Tu, R.S. Petralia, J.P. Yuan, A. Doan, C.D. Breder, A. Ruggiero, A.A. Lanahan, R.J. Wenthold, and P.F. Worley. 1998. Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins. Neuron. 21:707716.[Medline]
Xiao, B., J.C. Tu, and P.F. Worley. 2000. Homer: a link between neural activity and glutamate receptor function. Curr. Opin. Neurobiol. 10:370374.[CrossRef][Medline]
Xu, X., W. Zeng, J. Diaz, and S. Muallem. 1996a. Spatial compartmentalization of Ca2+ signaling complexes in pancreatic acini. J. Biol. Chem. 271:2468424690.
Xu, X., W. Zeng, and S. Muallem. 1996b. Regulation of the inositol 1,4,5-trisphosphate-activated Ca2+ channel by activation of G proteins. J. Biol. Chem. 271:1173711744.
Xu, X., W. Zeng, S. Popov, D.M. Berman, I. Davignon, K. Yu, D. Yowe, S. Offermanns, S. Muallem, and T.M. Wilkie. 1999. RGS proteins determine signaling specificity of Gq-coupled receptors. J. Biol. Chem. 274:35493556.
Zeng, W., X. Xu, and S. Muallem. 1996. Gß transduces [Ca2+]i oscillations and G
q a sustained response during stimulation of pancreatic acinar cells with [Ca2+]i-mobilizing agonists. J. Biol. Chem. 271:1852018526.
Zeng, W., X. Xu, S. Popov, S. Mukhopadhyay, P. Chidiac, J. Swistok, W. Danho, K.A. Yagaloff, S.L. Fisher, E.M. Ross, et al. 1998. The N-terminal domain of RGS4 confers receptor-selective inhibition of G protein signaling. J. Biol. Chem. 273:3468734690.
Zhao, X.S., D.M. Shin, L.H. Liu, G.E. Shull, and S. Muallem. 2001. Plasticity and adaptation of Ca2+ signaling and Ca2+-dependent exocytosis in SERCA2+/- mice. EMBO J. 20:26802689.