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J. Biol. Chem., Vol. 280, Issue 45, 37974-37987, November 11, 2005
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Epidermal Growth Factor Induces Tyrosine Phosphorylation, Membrane Insertion, and Activation of Transient Receptor Potential Channel 4*
From the School of Biomedical Sciences, Level 5 MSB, University of Newcastle, University Drive, Callaghan, New South Wales 2308, Australia
Various members of the canonical family of transient receptor potential channels (TRPCs) exhibit increased cation influx following receptor stimulation or Ca2+ store depletion. Tyrosine phosphorylation of TRP family members also results in increased channel activity; however, the link between the two events is unclear. We report that two tyrosine residues in the C terminus of human TRPC4 (hTRPC4), Tyr-959 and Tyr-972, are phosphorylated following epidermal growth factor (EGF) receptor stimulation of COS-7 cells. This phosphorylation was mediated by Src family tyrosine kinases (STKs), with Fyn appearing to be the dominant kinase. In addition, EGF receptor stimulation induced the exocytotic insertion of hTRPC4 into the plasma membrane dependent on the activity of STKs and was accompanied by a phosphorylation-dependent increase in the association of hTRPC4 with Na+/H+ exchanger regulatory factor. Furthermore, this translocation and association was defective upon mutation of Tyr-959 and Tyr-972 to phenylalanine. Significantly, inhibition of STKs was concomitant with a reduction in Ca2+ influx in both native COS-7 cells and hTRPC4-expressing HEK293 cells, with cells expressing the Y959F/Y972F mutant exhibiting a reduced EGF response. These findings represent the first demonstration of a mechanism for phosphorylation to modulate TRPC channel function.
Received for publication, April 4, 2005 , and in revised form, August 29, 2005.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 To whom correspondence should be addressed. Tel.: 61-2-4921-7827; Fax: 61-2-4921-7406: E-mail: adam.odell{at}newcastle.edu.au.
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