RhoA function in lamellae formation and migration is regulated by the α6β4 integrin and cAMP metabolism

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 alpha 6 beta 4 integrin. Here, we report that expression of a dominant negative RhoA (N19RhoA) in clone A cells inhibited alpha 6 beta 4-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 alpha 6 beta 4 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 beta 1 integrins, however, actually suppressed Rho A activation. The alpha 6 beta 4-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 beta 1 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.