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J. Biol. Chem., Vol. 282, Issue 11, 8474-8486, March 16, 2007

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Membrane-permeable Calmodulin Inhibitors (e.g. W-7/W-13) Bind to Membranes, Changing the Electrostatic Surface Potential

DUAL EFFECT OF W-13 ON EPIDERMAL GROWTH FACTOR RECEPTOR ACTIVATION^*

Parijat Sengupta, María José Ruano, Francesc Tebar^¶1, Urszula Golebiewska, Irina Zaitseva, Carlos Enrich^¶, Stuart McLaughlin², and Antonio Villalobo³

From the Department of Physiology and Biophysics, Health Science Center, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científica and Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain, and the ^¶Departament de Biologia Cellular i Anatomia Patològica, Universitat de Barcelona, Facultat de Medicina, Casanova 143, E-08036 Barcelona, Spain

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca²⁺/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca²⁺/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca²⁺/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.

Received for publication, July 28, 2006 , and in revised form, December 15, 2006.

^* This work was supported by National Institutes of Health Grant GM24971 and a grant from the Baldwin Foundation (to S. M.); Dirección General de Investigación, Ministerio de Educación y Ciencia Grant SAF2005-00631, Fondo de Investigaciones Sanitarias Grant RTICCC C03/10, and European Commission Grant MRTN-CT-2005-019561 (to A. V.); Ministerio de Educación y Ciencia Grants BMC2003-04754 and GEN2003-20662) (to C. E.); and Ministerio de Educación y Ciencia Grant BMC2003-09496 (to F. T.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ Supported by the Ramón y Cajal research Program, Ministerio de Educación y Ciencia (Spain).

² To whom correspondence may be addressed. Tel.: 631-444-3615; Fax: 631-444-3432; E-mail: Stuart.McLaughlin{at}stonybrook.edu.

³ To whom correspondence may be addressed. Tel.: 34-91-585-4424; Fax: 34-91-585-4401; E-mail: antonio.villalobo{at}iib.uam.es.

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