Extracellular Ca(2+) (Cao(2+))-induced E-cadherin-mediated cell-cell adhesion plays a critical role in promoting differentiation in epidermal keratinocytes. Our previous studies show that the calcium-sensing receptor (CaR) regulates keratinocyte cell-cell adhesion and differentiation via Rho A-mediated signaling. CaR forms a protein complex with Rho A, guanine nucleotide exchange factor Trio, and a cytoskeletal actin-binding protein, filamin A, at the cell-cell junctions in response to elevated Cao(2+) levels. Filamin A has the ability to interact directly with CaR, Trio, and Rho and mediate CaR-dependent signaling events.This study was conducted to investigate the roles of filamin A and Trio in regulating Cao(2+)-induced Rho activation and intercellular adhesion.Expression of filamin A and Trio in keratinocytes was inhibited by siRNA. Its effects on Cao(2+)-dependent junction formation and adhesion complex formation were evaluated by fluorescence immunostaining and immunoprecipitation. Endogenous Rho activity and expression of keratinocyte differentiation markers were also examined. The significance of the physical interactions of filamin A with Trio and Rho was assessed in dominant-negative inhibition studies.Inhibiting filamin A expression blocked the formation of CaR-Rho A-Trio-E-cadherin protein complex. Knockdown of filamin A or Trio inhibited Cao(2+)-induced membrane localization and activation of Rho A, formation of the E-cadherin-catenin adhesion complex, and keratinocyte terminal differentiation. Expressing dominant-negative peptides disruptive to the endogenous filamin-Trio, filamin-Rho, and CaR-filamin interactions suppressed the formation of adherens junctions.Through physical interactions with CaR, Trio and Rho, filamin A generates a scaffold for organizing a signaling complex that promotes E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation.