Obligatory Role of Src Kinase in the Signaling Mechanism for TRPC3 Cation Channels

Members of the canonical transient receptor potential (TRPC) subfamily of cation channels are candidates for capacitative and non-capacitative Ca2+ entry channels. When ectopically expressed in cell lines, TRPC3 can be activated by phospholipase C-mediated generation of diacylglycerol or by addition of synthetic diacylglycerols, independently of Ca2+ store depletion. Apart from this mode of regulation, little is known about other receptor-dependent signaling events that modulate TRPC3 activity. In the present study the role of tyrosine kinases in receptor- and diacylglycerol-dependent activation of TRPC3 was investigated. In HEK293 cells stably expressing TRPC3, pharmacological inhibition of tyrosine kinases, and specifically of Src kinases, abolished activation of TRPC3 by muscarinic receptor stimulation and by diacylglycerol. Channel regulation was lost following expression of a dominant-negative mutant of Src, or when TRPC3 was expressed in an Src-deficient cell line. In both instances, wild-type Src restored TRPC3 regulation. We conclude that Src plays an obligatory role in the mechanism for receptor and diacylglycerol activation of TRPC3. Members of the canonical transient receptor potential (TRPC) subfamily of cation channels are candidates for capacitative and non-capacitative Ca2+ entry channels. When ectopically expressed in cell lines, TRPC3 can be activated by phospholipase C-mediated generation of diacylglycerol or by addition of synthetic diacylglycerols, independently of Ca2+ store depletion. Apart from this mode of regulation, little is known about other receptor-dependent signaling events that modulate TRPC3 activity. In the present study the role of tyrosine kinases in receptor- and diacylglycerol-dependent activation of TRPC3 was investigated. In HEK293 cells stably expressing TRPC3, pharmacological inhibition of tyrosine kinases, and specifically of Src kinases, abolished activation of TRPC3 by muscarinic receptor stimulation and by diacylglycerol. Channel regulation was lost following expression of a dominant-negative mutant of Src, or when TRPC3 was expressed in an Src-deficient cell line. In both instances, wild-type Src restored TRPC3 regulation. We conclude that Src plays an obligatory role in the mechanism for receptor and diacylglycerol activation of TRPC3. In many types of cells, membrane receptors coupled to phospholipase C (PLC) 1The abbreviations used are: PLC, phospholipase C; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol; OAG, 1-oleoyl-2-acetyl-sn-glycerol; TRP, transient receptor potential; TRPC, canonical transient receptor potential; TRPM, melastatin-related transient potential; TRPV, vanilloid receptor-related transient receptor potential; GPCR, G-protein-coupled receptor; RTK, receptor tyrosine kinase; HBSS, Hepes-buffered saline solution; PKC, protein kinase C; HA, hemagglutinin; GFP, green fluorescence protein; WT, wild-type; DN, dominant-negative; EYFP, enhanced yellow fluorescent protein; EGF, epidermal growth factor; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo3,4-d-pyrimidine; bpV(phen), bisperoxo(1,10-phenantroline)oxovanadate; BCR, B cell receptor. promote inositol 1,4,5-trisphosphate (IP3)-mediated release of Ca2+ from endoplasmic reticulum and Ca2+ entry across the plasma membrane through both capacitative or store-operated and non-capacitative calcium entry pathways (1Putney Jr., J.W. Cell Calcium. 1986; 7: 1-12Crossref PubMed Scopus (2108) Google Scholar, 2Broad L.M. Cannon T.R. Taylor C.W. J. Physiol. (Lond.). 1999; 517: 121-134Crossref Scopus (195) Google Scholar, 3Shuttleworth T.J. Thompson J.L. Biochem. J. 1996; 316: 819-824Crossref PubMed Scopus (50) Google Scholar). Although the molecular identity of capacitative and non-capacitative calcium entry channels, as well as the signaling mechanisms involved, remain uncertain, mammalian homologues of the Drosophila melanogaster transient receptor potential (TRP) channel have been considered as potential candidates (4Birnbaumer L. Zhu X. Jiang M. Boulay G. Peyton M. Vannier B. Brown D. Platano D. Sadeghi H. Stefani E. Birnbaumer M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15195-15202Crossref PubMed Scopus (356) Google Scholar, 5Zitt C. Halaszovich C.R. Lückhoff A. Prog. Neurobiol. 2002; 66: 243-264Crossref PubMed Scopus (125) Google Scholar), particularly members of the canonical TRP (TRPC) subfamily (designated TRPC1 through TRPC7 (6Montell C. Birnbaumer L. Flockerzi V. Bindels R.J. Bruford E.A. Caterina M.J. Clapham D.E. Harteneck C. Heller S. Julius D. Kojima I. Mori Y. Penner R. Prawitt D. Scharenberg A.M. Schultz G. Shimizu N. Zhu M.X. Mol. Cell. 2002; 9: 229-231Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar)). The human isoform of TRPC3, originally cloned by Zhu et al. (7Zhu X. Jiang M. Peyton M. Boulay G. Hurst R. Stefani E. Birnbaumer L. Cell. 1996; 85: 661-671Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar), has been shown in many heterologous expression systems to behave as a receptor-activated channel that can be activated also by exogenous application of diacylglycerols (DAGs) independently of store depletion. In fact, DAG-induced activation of TRPC3 and its structural relatives TRPC6 and TRPC7 (8Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-262Crossref PubMed Scopus (1254) Google Scholar, 9Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar) provides the likely mechanism of activation of these channels by phosphoinositide-specific PLC-linked receptors, independently of IP3 and store depletion. Apart from its regulation by DAG generated from PLC stimulation through either G-protein-coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs), little is known about additional signaling pathways downstream of receptor-stimulation involved in activation of TRPC3. Stimulation of either of these receptor pathways results in rapid tyrosine phosphorylation of cellular proteins (10Tsai W. Morielli A.D. Peralta E.G. EMBO J. 1997; 16: 4597-4605Crossref PubMed Scopus (188) Google Scholar, 11Babnigg G. Zagranichnaya T. Wu X. Villereal M.L. J. Biol. Chem. 2003; 278: 14872-14882Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar), and so in the present study we investigated whether tyrosine kinases might play a role in the signaling mechanism underlying receptor- and DAG-dependent activation of TRPC3. We found that inhibition of tyrosine kinase activity completely abrogated the ability of either GPCR stimulation or exogenous application of diacylglycerol to induce TRPC3 activation. The results indicate an obligatory requirement for functional Src kinase for TRPC3 activation, likely involving Src-dependent phosphorylation and/or recruitment of an as yet unidentified accessory protein. In addition, the results show that activation of the channels by diacylglycerol is unlikely to involve a direct action of diacylglycerol on the channels. Cell Culture, Transfection, and Measurement of Intracellular Calcium—Human embryonic kidney cells (HEK293) stably expressing the human TRPC3 (TRPC3-HEK293) with either the hemagglutinin (HA)-epitope (TRPC3-HA) or the green fluorescent protein (TRPC3-GFP) fused to its C terminus, were cultured as previously described (12McKay R.R. Szmeczek-Seay C.L. Lièvremont J.-P. St John Bird G. Zitt C. Jüngling E. Lückhoff A. Putney Jr., J.W. Biochem. J. 2000; 351: 735-746Crossref PubMed Scopus (113) Google Scholar, 13Wedel B.J. Vazquez G. McKay R.R. St John Bird G. Putney Jr., J.W. J. Biol. Chem. 2003; 278: 25758-25765Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). For transient transfections, either TRPC3-HA or TRPC3-GFP cells were grown in 6-well plates and transfected as indicated with 1 μg of either wild-type Src (WT-Src, kindly provided by Dr. Thomas E. Smithgall, Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, PA), a dominant-negative mutant of Src (DN-Src, kindly provided by Dr. Sara Courtneidge, Van Andel Research Institute, Grand Rapids, MI), a of Src of Dr. John of Research or the and as a by following the cells were and an additional used for calcium fluorescence with cells were as in et al. M. St John Bird G. McKay R.R. Birnbaumer L. Putney Jr., J.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). When Ca2+ was in a are In instances, when involved of fluorescence with by that with are The B cell and the mutant in were kindly provided by Dr. Kurosaki of Molecular cells with the Ca2+ and of Ca2+ were as described previously G. B.J. M. John Bird G. Putney Jr., J.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). of the wild-type and cells were by with either the human isoform of TRPC3 TRPC3 provided by Dr. the human muscarinic receptor in transfected or a of with as for were used for fluorescence and and cells were from the and cultured in For transient transfections, and cells grown in 6-well plates were transfected by following the with either human TRPC3 human muscarinic receptor transfected or a of with as for In either or was with TRPC3, and cells were and an additional used for calcium as described for HEK293 In involving transfected cells, fluorescence were the indicated with cells, by fluorescence In involving transient transfections, from cells are shown for a for stably transfected cells for the pharmacological of are are of the cells are and behave cell as an The of with independently that the In instances, is on the of when of cells either or show a when for of cells a Ca2+ of cells in the of an of of or of Ca2+ or entry were a for or of for or When the of was found to be was by are as Cell and and HEK293 cells were cells were in saline in 1 and in the for with either or the cells were with phosphate-buffered saline and in 1 1 1 1 1 1 on with were for in an and were used for with the The were with μg of or and of protein for were with for and and in and on from were and to with the indicated with the were by following the For of tyrosine phosphorylation of cellular cells were and as cells were for with or from cell were to and with of Src cells were in HBSS, in the for and with either or either in the or of calcium Ca2+ and in the of the protein kinase C was to the to The cells were with and as The were with μg of and of protein for were with for and and as for was a Src Zhu H. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). with the was by was from Molecular OAG, and were from human epidermal growth was from The was from and were from were from other used were of of and for and are indicated in the We the of inhibition of cellular tyrosine kinases on receptor-dependent TRPC3 activation by of cells as indicated and M. St John Bird G. McKay R.R. Birnbaumer L. Putney Jr., J.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). in in the was used these to the capacitative calcium entry L.M. Cannon T.R. Taylor C.W. J. Physiol. (Lond.). 1999; 517: 121-134Crossref Scopus (195) Google Scholar, M. St John Bird G. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in muscarinic stimulation of cells in in a transient release of Ca2+ as a of from of Ca2+ to the in a Ca2+ activation of TRPC3. a in TRPC3 activation, with inhibition The a tyrosine kinase to K. 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Google Scholar) in a from inhibition of TRPC3 activation of from cell for tyrosine the of tyrosine phosphorylation by is a of kinase we that a of Src activity in the of can by by the tyrosine these that receptor- and activation of TRPC3 tyrosine kinase activity. the identity of the tyrosine involved in receptor-dependent activation of TRPC3, a of for of tyrosine kinases was The potential of epidermal growth kinase and kinases, and the tyrosine kinase known to be activated downstream of stimulation of the muscarinic receptor T. Biochem. PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, H. Y. R.R. D. J. Physiol. 2003; PubMed Scopus Google Scholar, C. J. Physiol. (Lond.). 1999; Scopus Google Scholar, J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), were of HEK293 cells with the of the epidermal growth N. Wu E. G. A.M. J. Biol. Chem. 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PubMed Scopus Google Scholar). shown in both muscarinic receptor- and cation entry were completely abrogated by of the wild-type of Src In the store-operated was by Ca2+ entry the of the store-operated Ca2+ entry was by in membrane potential that might the for Ca2+ expression in HEK293 cells stably expressing TRPC3 to (TRPC3-GFP) that of the mutant either expression or the membrane of the channel of in cells with both and activation of TRPC3 of and was by of the indicated that of both were expressed and that the of protein expressed with the was that for expression of an additional to the that Src activity is obligatory for signaling to TRPC3, we expressed TRPC3 with the muscarinic and is a cell functional in both of the Src kinases C. EMBO J. 1999; PubMed Scopus Google Scholar). shown in addition of to the in the of a cation entry in many transfected cells to TRPC3 was in cells transfected with the receptor activation of the muscarinic in a transient in Ca2+ to release from the of an of TRPC3 was the cation entry to TRPC3 the was to these entry was and was of TRPC3 activity to entry these the of TRPC3 regulation in these cells was in to the of Src expression was by TRPC3 was with the muscarinic receptor and cells, were by stably the wild-type of Src cells C. 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Vazquez G. McKay R.R. St John Bird G. Putney Jr., J.W. J. Biol. Chem. 2003; 278: 25758-25765Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) were for to either the muscarinic or epidermal growth cells were and tyrosine phosphorylation of TRPC3 was by the We found these The by of also in a We Src activation by a specifically Src a of Src activation Zhu H. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We that Src was activated following stimulation of the muscarinic with the pharmacological a role for Src in activation of TRPC3, also activation of Src were to induce activation of Src when calcium was with the completely the ability of the DAG to induce activation of Src with a mechanism L.M. Y. R.J. Cell Biol. 1999; PubMed Scopus Google Scholar). results indicated that and activation of Src is unlikely to be to ability to TRPC3, a known to be of both calcium and activity. Src kinases with and proteins through tyrosine T. H. T. S. T. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, V. J. Biol. Chem. Full Text PDF PubMed Google Scholar), we for of TRPC3 with Src by the results from with both and were for of the channel with Src We the as to whether activation of Src TRPC3 in the of stimulation of DAG were HEK293 cells were transfected with a of Src and the activity of TRPC3 (12McKay R.R. Szmeczek-Seay C.L. Lièvremont J.-P. St John Bird G. Zitt C. Jüngling E. Lückhoff A. Putney Jr., J.W. Biochem. J. 2000; 351: 735-746Crossref PubMed Scopus (113) Google Scholar, X. Jiang M. Birnbaumer L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) was and with that from both and was used as a for and the following of entry was M. St John Bird G. McKay R.R. Putney Jr., J.W. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). TRPC3 activity and for and with that of or cells and that Src by was to the The of to activity is with the that the expression or of TRPC3. TRPC3 is expressed and channels are present in the plasma this that inhibition of Src activity the activation mechanism of TRPC3, the expression or of the channels. of the Src was by to the ability of Src to on the channels independently involved of a B cell cells to B cell receptor stimulation with activation of tyrosine kinases, Src kinases, are to IP3 and DAG the phosphoinositide-specific coupled to activation M. Y. Kurosaki T. J. PubMed Scopus Google Scholar, T. Maeda A. M. A. K. M. 2000; PubMed Scopus Google Scholar). expression of human TRPC3 in the wild-type cell a channel that can be activated through either stimulation or muscarinic receptor stimulation G. B.J. M. John Bird G. Putney Jr., J.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the shown in TRPC3 was and entry was stimulation of cells to Ca2+ release from stimulation in activation of TRPC3 in with the receptor-dependent activation of the channel when expressed in wild-type cells shown and G. B.J. M. John Bird G. Putney Jr., J.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). results indicated an requirement of expression for activation of TRPC3, likely to the requirement for DAG generation for channel activity. Src activation is known to be in these cells, this that Src activation is to TRPC3 channels. In of this stimulation of cells with the muscarinic receptor and TRPC3, in rapid release of Ca2+ from by activation of entry was in cells transfected with the receptor as in cells in G. B.J. M. John Bird G. Putney Jr., J.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), Ca2+ in cells was of the DAG on cation entry in in HEK293 cells, both muscarinic and activation of the channel in cells were completely by with and with from cells expressing and indicate Src kinase activity to be for channel activation, is in the of a DAG to channel activation. of the members of the canonical TRP the signaling mechanism underlying TRPC3 activation and regulation In many cell expression of TRPC3 a receptor-activated channel that can also be activated by exogenous application of diacylglycerols by diacylglycerols is also a of the other members of the TRPC6 and TRPC7 (8Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-262Crossref PubMed Scopus (1254) Google Scholar, 9Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. Chem. 1999; 274: 27359-27370Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), and provides a potential mechanism for activation of these channels by PLC-linked receptors, independently of IP3 and Ca2+ store depletion. Although DAG generation through stimulation of either or is as a mechanism for TRPC3 activation, little is known about additional signaling pathways of receptor stimulation involved in regulation of TRPC3 activity. the of potential phosphorylation in the TRPC3 are specifically the role of kinases in TRPC3 activity. provided that M. St John Bird G. McKay R.R. Birnbaumer L. Putney Jr., J.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) and protein kinase TRPC3 Y. X. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). HEK293 cells stably expressing TRPC3 we found that the tyrosine kinase Src is for signaling to TRPC3, whether through receptor activation, or through application of of this of the cells with a of Src, of other tyrosine kinases, abrogated both receptor- and activation of TRPC3. transient expression of a dominant-negative of Src cells completely both receptor- and TRPC3 activation. The store-operated was by that the requirement for Src activity was for signaling to TRPC3. that this is with the by other that Src plays a role in the store-operated E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Biochem. J. 2003; PubMed Scopus Google Scholar, G. Villereal M.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). was in a dominant-negative action by Src was by the by the wild-type of expression of TRPC3 cells and in a channel with the activity for TRPC3 of regulation by either receptor activation or when TRPC3 was expressed and cells, Src C. 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The action of Src to involve direct or tyrosine phosphorylation of TRPC3 protein. we that direct phosphorylation of TRPC3 might that might be transient and of and that might be by the used of TRPC3 to Src, and of Src to TRPC3, the proteins in the is in of channel protein phosphorylation and protein might be to specifically this et al. Kurosaki T. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) the of receptor or muscarinic receptor stimulation to entry in as of a role of in cation entry regulation. the of stimulation to TRPC3 when expressed in the cell indicate a requirement for DAG generation for channel activity (8Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-262Crossref PubMed Scopus (1254) Google Scholar, M. St John Bird G. McKay R.R. Birnbaumer L. Putney Jr., J.W. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, K. J. Biol. Chem. 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