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CELLULAR AND MOLECULAR
The Medical and Research Services of the Ralph H. Johnson Veterans Affairs Medical Center, and Department of Medicine (Nephrology Division) of the Medical University of South Carolina, Charleston, South Carolina
Received August 16, 2006; accepted October 17, 2006.
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Gi2
and/or Gi3 Jak2 activation tyrosine phosphorylation of CaM increased binding of CaM to NHE-1 induction of a conformational change in NHE-1 that unmasks an obscured proton-sensing and/or proton-transporting region of NHE-1 activation of NHE-1. The Gi/o-coupled 5-HT1A receptor now joins a handful of Gq-coupled receptors and hypertonic shock as upstream activators of this emerging pathway. In the course of this work, we have presented clear evidence that CaM can be activated through tyrosine phosphorylation in the absence of a significant role for elevated intracellular Ca2+. We have also shown for the first time that the association of CaM with NHE-1 in living cells is a dynamic process.
; Grinstein et al., 1989). NHE-1 also plays cell-specific roles in the development and maintenance of a transformed cellular phenotype, differentiation of some cell types, structural anchoring and cytoskeletal organization, bone resorption, cell cycle control, apoptosis, and a host of other cellular functions (Putney et al., 2002; Fliegel, 2005). NHE-1 has also been implicated in clinically relevant conditions such as hypertension (Garciandia et al., 1995), left ventricular hypertrophy (Karmazyn et al., 2003), and ischemia-reperfusion injury (Wang et al., 2003). Despite its ubiquitous expression in mammalian cells and its potential clinical relevance, much remains to be learned regarding the molecular mechanisms through which this important protein is regulated.
The structure of NHE-1 suggests that its regulation can occur through at least four mechanisms: 1) interaction of regulatory factor(s) (proteins or lipids) with the cytoplasmic carboxyl terminal region of NHE-1; 2) phosphorylation of serines, threonines, and/or tyrosines located in the cytoplasmic domains of NHE-1; 3) phosphorylation of regulatory factors; and/or 4) binding of Ca2+/calmodulin (CaM) to NHE-1 (Counillon and Pouyssegur, 2000). Rapid activation of NHE-1 by mitogens is typically associated with increases in its phosphorylation (Sardet et al., 1991). A variety of protein kinases has been suggested as candidates to regulate NHE-1, including protein kinase C (Sauvage et al., 2000), CaM-dependent kinase (Fliegel et al., 1992), myosin light chain kinase (Shrode et al., 1995), p160 Rho-associated kinase (Tominaga et al., 1998), phosphatidylinositol 3'-kinase (Sauvage et al., 2000), the Nck-interacting kinase (Yan et al., 2001), and members of the mitogen-activated protein kinase family (Takahashi et al., 1999). In vascular smooth muscle cells, the 90-kDa S6 kinase (p90rsk) can directly phosphorylate NHE-1 on Ser-703 and mediate an increase in Na+-H+ exchange in vivo (Takahashi et al., 1999). However, only half of the response to growth factors is eliminated after deletion of amino acids 636 through 815 of NHE-1 (the region with most of the potential phosphorylation sites), indicating that direct phosphorylation of NHE-1 is not essential for its regulation (Wakabayashi et al., 1994b).
Because direct phosphorylation of NHE-1 accounts for only a part of its regulation, we have become interested in alternative pathways of activation, with particular reference to the role of CaM. CaM is a ubiquitous intracellular Ca2+ receptor and Ca2+-binding protein. It is a member of the superfamily of EF-hand proteins. CaM has four EF-hand motifs, each of which is composed of two -helices connected by a 12-amino acid loop. When intracellular Ca2+ levels increase to the low micromolar range, all four EF-hands bind Ca2+, inducing a conformational change that results in binding to various target proteins (Crivici and Ikura, 1995). CaM is classically activated by increases in intracellular Ca2+, resulting in conformational changes in CaM and activation of target proteins. However, other mechanisms of regulating CaM are possible, albeit poorly understood. Primary among the alternate mechanisms of activating CaM is phosphorylation of CaM on serine-threonine or tyrosine residues. In that regard, we recently described a novel pathway for the activation of NHE-1 by Janus kinase 2 (Jak2) and CaM, through which Jak2-induced phosphorylation of CaM is required for activation of NHE-1 by Gq-coupled receptors and hypertonic medium (Mukhin et al., 2001; Garnovskaya et al., 2003a,b). Those findings suggest that Gq-coupled receptors and hypertonic medium stimulate NHE-1 through this pathway: stimulus Jak2 activation tyrosine phosphorylation of CaM binding of CaM to NHE-1 activation of NHE-1. In the current article, we explore the possibility that a prototypical Gi-coupled receptor, the serotonin 5-hydroxytryptamine1A (5-HT1A) receptor, also uses this emerging pathway to activate NHE-1.
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, 1993).
Cell Culture. Chinese hamster ovary (CHO)-K1 cells expressing 
50 fmol of 5-HT1A receptors/mg of protein (CHO-5-HT1AR cells) were maintained in Ham's F-12 medium, supplemented with 10% fetal calf serum, streptomycin (100 µg/ml), penicillin (100 units/ml), and gentamycin (400 µg/ml) at 37°C in a 5% CO2-enriched, humidified atmosphere. Twenty-four to 48 h before each experiment, cells were switched to serum-free medium containing 0.5% bovine serum albumin (BSA) (Sigma).
Microphysiometry. The microphysiometer uses a light addressable silicon sensor to detect extracellular protons (McConnell et al., 1992). Each of eight channels has two inlet ports for buffers, one of which usually contains a vehicle control, and the other of which carries the test substance. The cells are perfused with buffer, and valve switches and stop-start cycles are controlled by a programmable computer. Acidification rate data are transformed by a personal computer running CytoSoft version 2.0 (Guava Technologies, Hayward, CA) and are presented as the extracellular acidification rate in microvolts per second, which roughly corresponds to millipH units per minute (Nernst equation). To facilitate comparison of data between two channels, values are expressed as a percentage of a baseline determined by computerized analysis of the five data points before exposure of the cell monolayers to a test substance.
All of the experiments were performed as described previously (Garnovskaya et al., 1997, 2003a,b). Cells were plated onto polycarbonate membranes (3-µm pore size, 12-µm size) at a density of 300,000 cells/insert the night before experimentation. After cells were attached to the membranes, they were growth-arrested in serum-free culture medium for 20 h before the experiment. The day of the study, cells were washed with serum-free, bicarbonate-free Ham's F-12 medium, placed into the microphysiometer chambers, and perfused at 37°C with the same medium or balanced salt solutions. For studies using inhibitors, cells were perfused for 15 min with tyrosine kinase inhibitors or CaM inhibitors before treatment with 8-OH-DPAT. For most studies, the pump cycle was set to perfuse cells for 60 s, followed by a 30-s "pump-off" phase, during which proton efflux was measured from seconds 6 through 28. Cells were exposed to the test agent for three or four cycles (270–360 s). Valve switches (to add or remove test agents) were performed at the middle of the pump cycle. Data points were then acquired every 90 s. The peak effect during stimulation was expressed as the percentage increase from baseline.
Immunoprecipitation. Experiments were performed as described previously (Mukhin et al., 2001; Garnovskaya et al., 2003b). Quiescent cell monolayers were treated with 100 nM 8-OH-DPAT or vehicle for 10 min. In some cases, cells were preincubated with 40 µM AG490 for 15 min before experimentation. After treatment with 8-OH-DPAT or vehicle for 10 min, cells were lysed in 1 ml/100-mm dish of radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% NP-40, 1 mM NaF, 1 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and pepstatin at 1 µg/ml each). Cell lysates were precleared by incubating with a protein A-agarose bead slurry for 30 min at 4°C. Precleared lysates (1 µg/µl total cell protein) were incubated with anti-Jak2/protein A-agarose or with other antibodies overnight (1:20 dilutions) at 4°C. Immunoprecipitates were captured by addition of protein A-agarose. The agarose beads were collected by centrifugation, washed three times with RIPA buffer, resuspended in 2x Laemmli sample buffer, boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis and subsequent immunoblot analysis.
Immunoblotting. The immunoblot protocol used for these studies was identical to that described previously by us (Garnovskaya et al., 2003a), except that the dilutions of the various antibodies followed the manufacturer's recommendations and that the blots were developed using Vistra ECF reagent (Amersham, Arlington Heights, IL). Dilutions used were murine anti-GFP (1:1000), goat anti-luciferase (1:1000), rabbit anti-Jak2 (1 µg/ml), rabbit anti-phospho-Stat3-pY705 (1:1000), mouse anti-CaM (1 µg/ml), rabbit anti-NHE-1 (2.5 µg/ml), and rabbit anti-Stat3 (1 µg/ml). In some cases, cells were preincubated with inhibitors for 15 min before experimentation. Cells were incubated with 100 nM 8-OH-DPAT or vehicle for 10 min.
Jak2 Phosphorylation Assay. Phosphorylation of Jak2 in response to 8-OH-DPAT was assessed using a Jak2 dual phosphospecific antibody (QCB). Quiescent cells were treated with 100 nM 8-OH-DPAT for 10 min and lysed in RIPA buffer. The lysates were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions with 4 to 20% precast gels (Invitrogen, Carlsbad, CA). After semidry transfer to polyvinylidene difluoride membranes, membranes were blocked with a Blotto buffer and incubated with the phospho-Jak2 antibody (0.5 µg/ml). After incubation with alkaline phosphatase-linked secondary antibodies, immunoreactive bands were visualized by a chemiluminescent method (CDP Star, New England Biolabs, Beverly, MA) using preflashed X-AR film (Eastman Kodak, Rochester, NY) and quantified using a GS-670 densitometer and Molecular Analyst software (Bio-Rad, Hercules, CA).
Immunofluorescence and Confocal Microscopy. Before experiments, CHO-5-HT1AR cells were plated in six-well plates on collagen I-coated coverslips at 1 x 105 cells/well, cultured for 24 h, and then transferred to serum-free Ham's F-12 medium supplemented with 0.5% BSA for 24 to 48 h. Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and fixed in PBS/4% paraformaldehyde for 30 min. Cells were then permeabilized with PBS/0.2% Triton X-100 for 5 min and incubated with PBS/2% BSA for 30 min to reduce nonspecific background staining. To visualize CaM, cells were first incubated with a mouse monoclonal anti-CaM antibody (1:10 dilution) overnight at 4°C. The coverslips were washed four times for 5 min each with PBS/2% BSA followed by incubation with a tetramethylrhodamine B isothiocyanate-labeled goat anti-mouse antibody (1:50 dilution) for 30 min at room temperature. Cells were then washed four times for 5 min each with PBS/2% BSA and then twice for 5 min each with PBS. Coverslips then were mounted on glass slides with Vectashield mounting medium.
Generation of Constructs for Bioluminescence Resonance Energy Transfer. We generated cDNA constructs in which CaM is tagged in tandem to luciferase by in-frame ligation into the mammalian expression vector pRL-CMV-Renilla reniformis luciferase (RLuc) (Promega). Fusions to the amino terminus of luciferase were termed RLuc-N1, and those to the carboxyl terminus of luciferase were termed RLuc-C1. Fusions of enhanced yellow fluorescent protein (eYFP) were created by tagging in tandem to the carboxyl terminus of NHE-1, or vice versa. This was accomplished by in-frame ligation to the yellow variant GFP-topaz vectors, pGFP-N1-topaz (fusion to the amino terminus of eYFP, eYFP-N1), or pGFP-C1-topaz (fusion to the carboxyl terminus of eYFP, eYP-C1) (Biosignal Packard, Montreal, Canada), with or without flexible linker. CHO cells were cotransfected with constructs, and the emissions of light from eYFP and luciferase were measured before and after stimulation with agonist as described previously (Turner et al., 2004; Turner and Raymond, 2005).
Bioluminescence Resonance Energy Transfer. To determine whether CaM and NHE-1 come into close proximity in intact cells, we used bioluminescence resonance energy transfer (BRET), a technique that detects close proximity of proteins using energy transfer between luminescent and fluorescent tags. A bioluminescent donor source (RLuc) can transfer energy to an acceptor fluorophore (yellow variant of Aequorea GFP, YFP) within a radius of approximately 50 Å, and this transfer is virtually undetectable at distances greater than 100 Å (Xu et al., 1999). CHO-5-HT1AR cells were transiently transfected as described previously (Garnovskaya et al., 1997) and cultured in Ham's F-12 media for 48 h to allow for protein expression. Cells were detached with PBS/1 mM EDTA and distributed into a 96-well plate at 1 x 105 cells/well. Fluorescence measurements were acquired using a Victor2 multilabel plate reader (PerkinElmer, Shelton, CT). In some cases, cells were incubated with 1 µM 8-OH-DPAT for 5 min. Coelenterazine was then added to a final concentration of 5 µM, and sequential measurements were made with filters at 460 ± 25 and 525 ± 25 nm. The BRET ratio was calculated as the ratio of light emitted at 525 nm (eYFP) over the light emitted at 460 nm (luciferase).
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, 1998). In Fig. 1, we used microphysiometry to show that activation of NHE-1 is sensitive to inhibitors of Jak2 and CaM. Figure 1A shows representative tracings demonstrating that a CaM inhibitor (1 µM ophiobolin A) reduces the activation of NHE-1 induced by the 5-HT1A receptor agonist 8-OH-DPAT (100 nM). Figure 1B shows the results of microphysiometry experiments in which a panel of CaM inhibitors was tested for effects on activation of NHE-1 by 100 nM 8-OH-DPAT. Because results obtained with pharmacological inhibitors should be interpreted with caution because of potential nonspecific or nonselective effects, we used a panel of diverse CaM inhibitors to probe the involvement of CaM in the activation of NHE-1 by 8-OH-DPAT. Five structurally distinct CaM inhibitors (5 µM calmidazolium, 1 µM ophiobolin A, 50 µM W-7, 10 µM fluphenazine, and 10 µM chlorpromazine) each markedly suppressed the peak activation of NHE-1 by 70 to 90%. These results suggest that CaM is involved in 5-HT1A receptor-mediated activation of NHE-1. Because one key mechanism through which CaM is activated involves elevation of intracellular Ca2+ levels (Crivici and Ikura, 1995), we tested the effect of 50 µM BAPTA-AM, a cell-permeable calcium chelator, on 8-OH-DPAT (100 nM)-induced activation of NHE-1. Figure 1B shows that BAPTA-AM had no effect on the activation of NHE-1, suggesting that an alternative pathway is responsible for activation of CaM. This notion is supported by the fact that these cells do not mobilize Ca2+ in response to 5-HT or 8-OH-DPAT in concentrations as high as 5 µM (data not shown).
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, 1995; De Frutos et al., 1997; Corti et al., 1999). Indeed, we have proposed that Gq-coupled receptors and hypertonicity activate CaM (at least in part) through its tyrosine phosphorylation (Mukhin et al., 2001; Garnovskaya et al., 2003a,b). Figure 1C shows that the broad-spectrum tyrosine kinase inhibitor genistein (100 µM) strongly attenuates the activation of NHE-1 by 100 nM 8-OH-DPAT. In contrast, an inactive congener of genistein (100 µM daidzein) had no effect. These results support the idea that a tyrosine kinase is specifically involved in the activation of NHE-1 by the 5-HT1A receptor. Figure 1D shows that the epidermal growth factor receptor kinase inhibitor AG1478 (10 µM) had no effect on 5-HT1A receptor-activated NHE-1. In contrast, the Jak2 tyrosine kinase inhibitor AG490 (40 µM) markedly suppressed NHE-1 activity induced by 100 nM 8-OH-DPAT. These results suggest that both CaM and Jak2 are involved in the activation of NHE-1 by the 5-HT1A receptor.
Roles of Jak2 in the Activation of NHE-1 by the 5-HT1A Receptor. Figure 2A shows that 100 nM 8-OH-DPAT induces an increase in phosphorylation of Stat3 (an important Jak2 substrate) and that this phosphorylation can be markedly attenuated by the selective 5-HT1A receptor antagonist S-UH-301 (1 µM). A similar degree of phosphorylation was induced by 100 nM 5-HT, the endogenous ligand of the 5-HT1A receptor (not shown). In addition, neither 5-HT nor 8-OH-DPAT induced Stat3 phosphorylation in parental CHO-K1 cells not transfected with the 5-HT1A receptor (not shown). Thus, the transfected 5-HT1A receptor mediates Stat3 phosphorylation. Because nearly all of the signals emanating from the 5-HT1A receptor 8-OH-DPAT are mediated by PTX-sensitive heterotrimeric G proteins (Gi/o), we tested the effects of overnight incubation with PTX (200 ng/ml) on the ability of 100 nM to increase Stat3 phosphorylation. Figure 2B shows that PTX completely eliminated 8-OH-DPAT-induced phosphorylation of Stat3. In aggregate, these results show that the 5-HT1A receptor, acting through Gi/o proteins, induces phosphorylation of Stat3, and thus activation of Jak2, in CHO-K1 cells.
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Figure 2C shows that 8-OH-DPAT induces a concentration-dependent increase in the phosphorylation of Stat3, with half-maximal stimulation being achieved at 30 nM 8-OH-DPAT. Figure 2D shows that the Jak2 inhibitor AG490 (40 µM) suppressed 8-OH-DPAT-induced Stat3 phosphorylation by more than 80%. Results obtained by the use of a single pharmacological inhibitor of a target enzyme should be confirmed using other compounds or methods. Thus, we compared the effects of AG490 with those of a highly selective Jak3 inhibitor (Jak3 inhibitor I, WHI-P131) (Goodman et al., 1998) on the ability of 100 nM 8-OH-DPAT to induce phosphorylation of Stat3. Figure 2E shows that AG490 suppressed Stat3 phosphorylation in a concentration-dependent manner by 60% at 25 µM and >100% at 100 µM. In contrast, WHI-P131 had no statistically significant effects at concentrations as high as 300 µM. These data show that the 5-HT1A receptor activates Jak2 and support the possibility that Jak2 plays a key role in the activation of NHE-1 by the 5-HT1A receptor.
One key pathway of activation of NHE-1 is by the formation of protein complexes containing CaM, which binds directly to the carboxyl terminus of NHE-1 and induces a conformational change that unmasks a proton-sensing region of NHE-1 (Wakabayashi et al., 1994a). We used coimmunoprecipitation to provide further evidence supporting a key role for Jak2 in the activation of NHE-1 by the 5-HT1A receptor. Figure 3A shows that the amount of CaM in phosphotyrosine immunoprecipitates was increased by 8-OH-DPAT and that the increase was attenuated by 70% by 40 µM AG490. Similar results were obtained for the converse study (n = 2; data not shown), in that the phosphotyrosine content of CaM was increased in response to 100 nM 8-OH-DPAT and that the increase in phosphotyrosine immunoreactivity was markedly attenuated by 40 µM AG490. Figure 3B shows that there was virtually no association of Jak2 and CaM in Jak2 immunoprecipitates derived from quiescent cells. When cells were stimulated with 100 nM 8-OH-DPAT, there was a large increase in CaM in the Jak2 immunoprecipitates. Moreover, AG490 nearly completely blocked this association. Figure 3C shows similar results when the CaM immunoprecipitates were probed with anti-Jak2 antibodies. We have shown previously that the 5-HT1A receptor binds constitutively to CaM. To assess whether Jak2 modulates that binding, we immunoprecipitated the 5-HT1A receptor using an antibody that we have previously characterized (Raymond et al., 1989, 1993), after which we probed for the presence of CaM. Figure 3D shows that CaM was associated with the 5-HT1A receptor under basal conditions and that this association was not significantly altered by treatment with 8-OH-DPAT or with AG490. These results suggest that Jak2 interacts with CaM downstream of the 5-HT1A receptor. Indeed, Fig. 3E shows that 8-OH-DPAT increases the amount of CaM in NHE-1 immunoprecipitates, an effect that was attenuated by 75% by AG490. This result suggests that Jak2 modulates the interaction of CaM with NHE-1. The results in Fig. 3F suggest that Jak2 is present in NHE-1 immunoprecipitates in cells treated with 8-OH-DPAT and that AG490 attenuates this interaction by 70%.
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The 5-HT1A Receptor Activates NHE-1 by Inducing a Direct Interaction of CaM with the NHE-1 Carboxyl Terminus. Although it is generally accepted that CaM activates NHE-1 by binding directly to its carboxyl terminus (Wakabayashi et al., 1994a), this idea has never been directly tested in living cells. Therefore, we tested this idea using BRET between CaM and the carboxyl terminus of NHE-1. cDNA encoding fusion protein constructs were expressed in CHO cells and probed by immunoblot with various antibodies to confirm expression. Figure 4 shows the results of expression of various constructs in CHO cells. Figure 4A shows experiments in which anti-GFP antibodies were used to identify CaM eYFP fusion proteins by immunoblot. Lanes 1 and 10 show immunoblots from lysates derived from mock-transfected CHO cells, and lane 11 shows nontransfected CHO cells. Lanes 2 and 3 show expression of control (nonfused) eYFP-N1 and eYFP-C1 constructs. Lanes 4, 5, 8, and 9 show luciferase fusions, which have no specific immunoreactivity with the GFP antibody. Lanes 6 and 7 show eYFP-N1-CaM and eYFP-C1-CaM, the sizes of which increase by 18 kDa, as would be expected for CaM fusions. Figure 4B shows results of immunoblots from the same lysates using an anti-luciferase antibody. Lanes 4 and 5 show control RLuc-N1 and RLuc-C1 constructs. Lanes 8 and 9 show RLuc-N1-CaM and RLuc-C1-CaM, the sizes of which increase by 18 kDa, as would be expected for CaM.
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We then used transient cotransfection of BRET pairs in CHO cells expressing the 5-HT1A receptor. Results were expressed as the normalized ratio of fluorescence to luminescence in the various wells. Figure 5A shows a positive control experiment in which RLuc alone resulted in little or no BRET signal. Cotransfection of RLuc and eYFP did not generate a BRET signal, indicating that the two molecules do not aggregate with each other to such an extent that they would yield a false-positive BRET signal. In contrast, when eYFP was fused either to the amino or carboxyl terminal end of RLuc, measurable signals resulted, with the carboxyl terminal fusion yielding a signal slightly greater than double that generated by the amino terminal fusion.
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Figure 5B shows that RLuc fused to NHE-1 (RLuc-N1-NHE-1) yielded virtually no signal. Importantly, when that construct was cotransfected along with eYFP, a small signal (<10 mBU, mean-specific BRET ratio value) resulted. This signal is probably the result of nonspecific interactions. This signal is relatively small and is less than 20% of that generated when RLuc-N1-NHE-1 was cotransfected with eYFP-N1-CaM. This suggests that CaM and NHE-1 can interact with each other (come within 100 Å of each other) to a much greater degree than background interactions between RLuc-NHE-1 and nonfused eYFP. To further support the specificity of the interaction between RLuc-N1-NHE-1 and eYFP-N1-CaM, we stimulated CHO-5-HT1AR cells with the selective receptor agonist 8-OH-DPAT. Treatment with 1 µM 8-OH-DPAT had no effect on the BRET signal in cells expressing RLuc-N1-NHE-1 plus nonfused eYFP, but 8-OH-DPAT treatment induced a 2.5-fold increase in the BRET signal in cells expressing RLuc-N1-NHE-1 plus eYFP-N1-CaM. Similar results were obtained when cells were transfected with RLuc-N1-CaM plus eYFP-N1-NHE-1, or with RLuc-C1-CaM plus eYFP-N1-NHE-1 (Fig. 5C), although the former BRET pair yielded a basal signal double that of the latter pair. In addition, 100 nM 8-OH-DPAT significantly increased the BRET signals for RLuc-N1-CaM plus eYFP-N1-NHE-1, or with RLuc-C1-CaM plus eYFP-N1-NHE-1. The increased BRET signal was significantly attenuated by 40 µM AG490 for both pairs of constructs (Fig. 5C), suggesting that Jak2 is involved in the pathway that induces CaM and NHE-1 to associate in live cells. AG490 did not significantly diminish the basal BRET signals (data not shown), suggesting that Jak2 is not involved in the constitutive association of CaM and NHE-1. Moreover, a Jak3 inhibitor (200 µM WHI-P131) had no effect (data not shown). These studies show that an agonist known to increase NHE-1 activity induces CaM and NHE-1 to come into proximity to each other without inducing nonfused eYFP to come into proximity with NHE-1. Moreover, the strength of the BRET signal depends on whether luciferase has been fused to the carboxyl or amino terminal end of CaM, as would be expected if the interaction of CaM with NHE-1 were selective, rather than nonspecific. The studies also strongly support the involvement of Jak2 in 5-HT1A receptor-induced association of CaM and NHE-1.
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The work is remarkable with regard to several issues. First, the 5-HT1A receptor now joins a handful of Gq-coupled receptors and hypertonic shock as upstream activators of this emerging pathway. Second, we have presented clear evidence that CaM can be activated through tyrosine phosphorylation in the absence of a significant role for elevated intracellular Ca2+. Third, we have shown for the first time that the association of CaM with NHE-1 in living cells is a dynamic process.
We have previously shown that the bradykinin B2, 5-HT2A, and angiotensin II AT1A receptors, all of which couple to Gq/11, can rapidly activate NHE-1 through a novel pathway that involves coordinated activities of CaM and Jak2 (Garnovskaya et al., 1998, 2003a). In addition, we have shown that exposure of cells to hypertonic media activates NHE-1 through a similar mechanism (Garnovskaya et al., 2003b). One key difference is that the Gq/11-coupled receptors engage this pathway to some extent through elevated intracellular Ca2+, whereas hypertonic medium engages this pathway independent of elevated intracellular Ca2+. The current study shows that the Gi/o-coupled 5-HT1A receptor also activates NHE-1 through CaM and Jak2. However, the pathway more closely resembles that induced by hypertonic exposure in that elevations of intracellular Ca2+ are not involved. Thus, the results show that CaM and Jak2 link Gi/o-coupled receptors, as well as Gq/11-coupled receptors and hypertonic medium, to NHE-1 activation. They also show that significant elevations of intracellular Ca2+ are not necessary for CaM to play a critical role in the activation of NHE-1.
CaM has been previously shown to be a substrate for phosphorylation by both tyrosine kinases and serine-threonine kinases (Sacks et al., 1992, 1995; Corti et al., 1999). Activation of the insulin receptor (a receptor tyrosine kinase) has been shown to result in phosphorylation of Tyr-99 and Tyr-138 of CaM in CHO-IR cells (De Frutos et al., 1997). The epidermal growth factor receptor (another receptor tyrosine kinase) phosphorylates Tyr-99 of bovine brain CaM (De Frutos et al., 1997; Benaim and Villalobo, 2002) with a stoichiometry of 1:1. In contrast, casein kinase II, an insulin-sensitive nonreceptor kinase, phosphorylates CaM in vitro on serine and threonine residues (Thr-79, Ser-81, Ser-101, and Thr-117) (Sacks et al., 1992). We recently showed that CaM could serve as a substrate for purified Jak2 (Mukhin et al., 2001), although the current work does not definitively show a direct role for Jak2 in the tyrosine phosphorylation of CaM after activation of the 5-HT1A receptor. It is noteworthy that both insulin and epidermal growth factor receptors have been shown to phosphorylate CaM on Tyr-99 and/or Tyr-138 of CaM (De Frutos et al., 1997; Benaim and Villalobo, 2002); these are the only tyrosine residues within CaM. Based on the crystal structure of CaM, Tyr-99 is located within the third Ca2+-binding domain and is somewhat more exposed than Tyr-138 (Benaim and Villalobo, 2002).
There is no consensus on the effects of phosphorylation of CaM on its ability to interact with and activate its downstream targets. Our work suggests that tyrosine phosphorylation of CaM results in increased binding to and activation of NHE-1. Unlike our studies, Fukami et al. (1986) previously suggested that Ca2+-induced phosphorylation of CaM may attenuate its function in vivo. Phosphorylation of CaM on Tyr-99 was shown to selectively attenuate the action of CaM antagonists on type 1 cyclic nucleotide phosphodiesterase activity (Saville and Houslay, 1994). In contrast, phosphorylation of Tyr-99 increases the affinity of CaM for Ca2+-ATPase (Sacks et al., 1996). To address that discrepancy, Corti et al. (1999) studied the effects of CaM phosphorylation on Tyr-99 on the binding affinities and activation of six different CaM target enzymes [myosin light chain kinase, 3'-5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, Ca2+-CaM-dependent protein phosphatase 2B (calcineurin), neuronal nitric-oxide synthase, and type 2 Ca2+-CaM-dependent protein kinase]. They concluded that tyrosine phosphorylation of CaM Tyr-99 generally led to an increase in the ability of CaM to activate its targets. For three of the enzymes (3'-5'-cyclic nucleotide phosphodiesterase, plasma membrane Ca2+-ATPase, and type 2 Ca2+-CaM-dependent protein kinase) the primary effect was a decrease in the concentration at which half-maximal activation was attained. In contrast, for calcineurin and neuronal nitric oxide synthase, phosphorylation of CaM significantly increased the Vmax. For myosin light chain kinase, however, tyrosine phosphorylation of CaM had no effect (Corti et al., 1999). Thus, the idea that tyrosine phosphorylation of CaM results in increased binding to and activation of NHE-1 is supported by precedent with other CaM-activated enzymes.
The idea that the activation of NHE-1 by CaM involves a conformational change in NHE-1 that unmasks its proton-sensing and/or transport region was supported by early mutagenesis work by Pouyssegur's group (Wakabayashi et al., 1994a,b, 1995, 2003). However, the direct observation of the dynamic interaction between NHE-1 and CaM in living cells has heretofore not been shown. Our work fills that gap in the knowledge base in that 5-HT1A receptor activation leads to an increased BRET signal between CaM and the carboxyl terminus of NHE-1. Clearly, considerable structural work remains to be done to fully test the ideas proposed by Pouyssegur's group. However, when taken together with our previous work (Garnovskaya et al., 1997) and that of Pouyssegur's group, the following pathway of activation of NHE-1 is probable: 5-HT1A receptor Gi2 and/or Gi3 Jak2 activation tyrosine phosphorylation of CaM increased binding of CaM to NHE-1 induction of a conformational change in NHE-1 that unmasks an obscured proton-sensing and/or proton-transporting region of NHE-1 activation of NHE-1.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NHE-1, type 1 sodium-proton exchanger; CaM, calcium/calmodulin; Jak2, Janus kinase 2; 5-HT1A, 5-hydroxytryptamine1A; W-7, N-(6-aminohexyl)5-chloro-1-naphthalene sulfonamide; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester; PTX, Pertussis toxin; AG490, N-benzyl-3,4-dihydroxy-benzylidenecyanoacetamide; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)-tetralin; Stat3, signal transducer and activator of transcription 3; GFP, green fluorescent protein; CHO, Chinese hamster ovary; BSA, bovine serum albumin; RIPA, radioimmunoprecipitation assay; PBS, phosphate-buffered saline; RLuc, Renilla reniformis luciferase; eYFP, enhanced yellow fluorescent protein; BRET, bioluminescence resonance energy transfer; genistein, 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one; daidzein, 7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one; AG1478, 4-(3-chloroanillino)-6,7-dimethoxyquinazoline; UH-301, 5-fluoro-8 hydroxy-2-(dipropylamino)-tetralin; ECAR, extracellular acidification rate.
Address correspondence to: Dr. John Raymond, 179 Ashley Avenue, Office of the Provost, Charleston, SC 29425. E-mail: raymondj{at}musc.edu
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