Abstract
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), Ca2+/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 → Gi2α and/or Gi3α → 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.
Footnotes
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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.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.112581.
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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.
- Received August 16, 2006.
- Accepted October 17, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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