Brief Report

RhoA Function in Lamellae Formation and Migration Is Regulated by the 6ß4 Integrin and cAMP Metabolism

Correspondence to: Arthur M. Mercurio, Beth Israel Deaconess Medical Center, Department of Pathology, Research North, 330 Brookline Avenue, Boston, MA 02215. Tel:(617) 667-7714 Fax:(617) 975-5531 E-mail:amercuri{at}caregroup.harvard.edu.

Clone A colon carcinoma cells develop fan-shaped lamellae and exhibit random migration when plated on laminin, processes that depend on the ligation of the 6ß4 integrin. Here, we report that expression of a dominant negative RhoA (N19RhoA) in clone A cells inhibited 6ß4-dependent membrane ruffling, lamellae formation, and migration. In contrast, expression of a dominant negative Rac (N17Rac1) had no effect on these processes. Using the Rhotekin binding assay to assess RhoA activation, we observed that engagement of 6ß4 by either antibody-mediated clustering or laminin attachment resulted in a two- to threefold increase in RhoA activation, compared with cells maintained in suspension or plated on collagen. Antibody-mediated clustering of ß1 integrins, however, actually suppressed Rho A activation. The 6ß4-mediated interaction of clone A cells with laminin promoted the translocation of RhoA from the cytosol to membrane ruffles at the edges of lamellae and promoted its colocalization with ß1 integrins, as assessed by immunofluorescence microscopy. In addition, RhoA translocation was blocked by inhibiting phosphodiesterase activity and enhanced by inhibiting the activity of cAMP-dependent protein kinase. Together, these results establish a specific integrin-mediated pathway of RhoA activation that is regulated by cAMP and that functions in lamellae formation and migration.

Key Words: carcinoma, protein kinase A, G-protein, phosphodiesterase, cytoskeleton

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