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Volume 272, Number 48, Issue of November 28, 1997 pp. 30115-30121
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Ileal Microvillar Protein Villin Is Tyrosine-phosphorylated and Associates with PLC-₁
ROLE OF CYTOSKELETAL REARRANGEMENT IN THE CARBACHOL-INDUCED INHIBITION OF ILEAL NaCl ABSORPTION

(Received for publication, July 30, 1997, and in revised form, September 22, 1997)

From the Departments of Medicine and Physiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the ^§ Institut Curie, Paris 75231, France

In ileal absorptive cells, carbachol inhibits NaCl absorption and its component brush border Na⁺/H⁺ exchanger, acting via basolateral membrane receptors. This carbachol effect involves (i) activation of brush border phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity and brush border but not basolateral membrane translocation of PLC-₁ (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, W. A., Rhee, S. G., Donowitz, M., and Cohen, M. E. (1996) Biochem. J. 313, 509-518); and (ii) brush border tyrosine kinase(s) because mucosal but not serosal addition of the tyrosine kinase inhibitor genistein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na⁺/H⁺ exchange. In the present work we identify a pool of villin (a brush border actin-binding protein) in the microvillus membrane fraction of rabbit ileum; this pool of villin is tyrosine-phosphorylated and associates with brush border membrane PLC-₁. Villin is present both in the Triton X-100-soluble and -insoluble fractions of the brush border. The Triton X-100-soluble pool is approximately 4-fold smaller than the brush border pool of villin that is present in the Triton X-100-insoluble fraction. Only the villin present in the Triton X-100-soluble fraction of ileal villus brush border associates with PLC-₁ and is tyrosine-phosphorylated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently. Carbachol also increases the amount of tyrosine-phosphorylated villin that associates with PLC-₁. These studies demonstrate that carbachol effects on NaCl absorption are accompanied by an increase in brush border PLC-₁ association with villin and an increase in tyrosine phosphorylation of villin. To study the role of cytoskeletal rearrangement in carbachol-induced inhibition of NaCl absorption, we used the F-actin stabilizing drug jasplakinolide. Jasplakinolide prevents the carbachol inhibition of ileal NaCl absorption. This suggests that F-actin severing is necessary for carbachol to inhibit ileal villus NaCl absorption. Since villin is known to sever actin, these studies suggest a role for villin in the signaling cascade that begins at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.

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