Ca -Calmodulin and Janus Kinase 2 Are Required for Activation of Sodium-Proton Exchange by the Gi-Coupled 5-Hydroxytryptamine1a Receptor

The type 1 sodium-proton exchanger (NHE-1) is expressed ubiquitously and regulates key cellular functions, including mitogenesis, cell volume, and intracellular pH. Despite its importance, the signaling pathways that regulate NHE-1 remain incompletely defined. In this work, we present evidence that stimulation of the 5-hydroxytryptamine1A (5-HT1A) receptor results in the formation of a signaling complex that includes activated Janus kinase 2 (Jak2), Ca /calmodulin (CaM), and NHE-1, and which involves tyrosine phosphorylation of CaM. The signaling pathway also involves rapid agonist-induced association of CaM and NHE-1 as assessed by coimmunoprecipitation studies and by bioluminescence resonance energy transfer studies in living cells. We propose that NHE-1 is activated through this pathway: 5-HT1A receptor 3 Gi2 and/or Gi3 3 Jak2 activation 3 tyrosine phosphorylation of CaM 3 increased binding of CaM to NHE-1 3 induction of a conformational change in NHE-1 that unmasks an obscured protonsensing and/or proton-transporting region of NHE-1 3 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 Ca . We have also shown for the first time that the association of CaM with NHE-1 in living cells is a dynamic process. The type 1 sodium-proton exchanger (NHE-1, also known as product of SLC9A1, solute carrier family 9A, type 1) is ubiquitous, being expressed on the plasma membrane of virtually every mammalian cell. It mediates the 1:1 exchange of extracellular Na for intracellular H , thereby maintaining intracellular pH (Pouyssegur et al., 1984; 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 This work was supported by grants from the Department of Veterans Affairs (Merit Awards and a REAP award to J.R.R. and M.N.G.), the National Institutes of Health (GM08716 to J.H.T.; DK52448 and GM63909 to J.R.R.), a predoctoral fellowship from the American Heart Association, Mid Atlantic Affiliate (0215195U to J.H.T.), and laboratory endowments jointly supported by the Medical University of South Carolina Division of Nephrology and Dialysis Clinics, Inc. (J.R.R.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.112581. 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. 0022-3565/07/3201-314–322 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 1 U.S. Government work not protected by U.S. copyright 112581/3164866 JPET 320:314–322, 2007 Printed in U.S.A. 314 at A PE T Jornals on July 7, 2017 jpet.asjournals.org D ow nladed from 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 Ca /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 (p90) 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 Ca receptor and Ca -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 Ca levels increase to the low micromolar range, all four EF-hands bind Ca , inducing a conformational change that results in binding to various target proteins (Crivici and Ikura, 1995). CaM is classically activated by increases in intracellular Ca , 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 3 Jak2 activation 3 tyrosine phosphorylation of CaM 3 binding of CaM to NHE-1 3 activation of NHE-1. In the current article, we explore the possibility that a prototypical Gi-coupled receptor, the serotonin 5-hydroxytryptamine1A (5HT1A) receptor, also uses this emerging pathway to activate NHE-1. Materials and Methods Materials. Calmidazolium, fluphenazine, chlorpromazine, W-7, ophiobolin A, BAPTA-AM, Pertussis toxin (PTX), and various salts were from Sigma (St. Louis, MO). AG490 was from Calbiochem (San Diego, CA). 8-OH-DPAT was from Sigma-Aldrich/RBI (Natick, MA). Anti-CaM monoclonal antibody, anti-Jak2 agarose-conjugated antibody, and anti-phosphotyrosine polyclonal antibody were from Upstate Biotechnology (Lake Placid, NY). Total signal transducer and activator of transcription 3 (Stat3) and anti-phosphospecific Stat3 (pY705) rabbit antibodies were from Biosource International (Camarillo, CA) or QCB (Hopkinton, MA). Anti-NHE-1 antibody was from Chemicon International (Temecula, CA). All of the cell culture media and supplements were from Life Sciences (Grand Island, NY). Polycarbonate cell culture inserts for microphysiometry were from Corning Costar (Cambridge, MA). The murine anti-green fluorescent protein (GFP) antibody was purchased from Clontech (Mountain View, CA). Goat polyclonal anti-luciferase antibody was obtained from Promega (Madison, WI). Anti-5-HT1A receptor rabbit serum was raised using a peptide sequence from the predicted third intracellular loop of the receptor (GASPAPQPKKKSVNGESGSRNWRLGVE) and thoroughly validated and characterized as described previously (Raymond et al., 1989, 1993). Cell Culture. Chinese hamster ovary (CHO)-K1 cells expressing 50 fmol of 5-HT1A receptors/mg of protein (CHO-5-HT R cells) were maintained in Ham’s F-12 medium, supplemented with 10% fetal calf serum, streptomycin (100 g/ml), penicillin (100 units/ml), and gentamycin (400 g/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 (3m pore size, 12m 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 M 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 g/ml each). Cell lysates were precleared by incubating with a protein A-agarose bead slurry for 30 Calmodulin, Jak2, and NHE-1 315 at A PE T Jornals on July 7, 2017 jpet.asjournals.org D ow nladed from min at 4°C. Precleared lysates (1 g/ l 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 2 Laemmli sample buffer, boiled for 5 min, and subjected to SDSpolyacrylamide gel electrophoresis and subsequent immunoblot