Abstract

Paracingulin is an M(r) 150-160 kDa cytoplasmic protein of vertebrate epithelial tight and adherens junctions and comprises globular head, coiled-coil rod, and globular tail domains. Unlike its homologous tight junction protein cingulin, paracingulin has been implicated in the control of junction assembly and has been localized at extrajunctional sites in association with actin filaments. Here we analyze the role of paracingulin domains, and specific regions within the head and rod domains, in the function and localization of paracingulin by inducible overexpression of exogenous proteins in epithelial Madin Darby canine kidney (MDCK) cells and by expression of mutated and chimeric constructs in Rat1 fibroblasts and MDCK cells. The overexpression of the rod + tail domains of paracingulin perturbs the development of the tight junction barrier and Rac1 activation during junction assembly by the calcium switch, indicating that regulation of junction assembly by paracingulin is mediated by these domains. Conversely, only constructs containing the head domain target to junctions in MDCK cells and Rat1 fibroblasts. Furthermore, expression of chimeric cingulin and paracingulin constructs in Rat1 fibroblasts and MDCK cells identifies specific sequences within the head and rod domains of paracingulin as critical for targeting to actin filaments and regulation of junction assembly, respectively. In summary, we characterize the functionally important domains of paracingulin that distinguish it from cingulin.

Highlights

  • Paracingulin is a junctional protein that regulates Rho GTPase activities in epithelial cells

  • Evidence from knockdown and knockout studies shows that neither protein is required to maintain the structural organization of the apical junctional complex (AJC) at steady state, both are required to down-regulate RhoA activity in confluent Madin Darby canine kidney (MDCK) cells, whereas paracingulin is required to promote Rac1 activation during junction assembly [26, 27, 31]

  • We show that the inducible overexpression of either full-length paracingulin or its domains does not result in the redistribution of tight junction (TJ) proteins or changes in TJ structure and barrier function at steady state

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Summary

Background

Paracingulin is a junctional protein that regulates Rho GTPase activities in epithelial cells. In vitro binding experiments with recombinant proteins indicate that regions both in the globular head and coiled-coil rod domains of paracingulin can interact directly with the RhoA and Rac guanidine exchange factors GEF-H1 and Tiam, providing a molecular mechanisms for the control of Rho GTPases and TJ assembly by paracingulin [26]. It is not known whether the head, the rod, or both domains regulate Rac1-dependent TJ assembly within cells. By examining the subcellular localization of these constructs in MDCK cells and Rat fibroblasts and junction assembly in MDCK cells, we define the roles of specific head and coiled-coil rod sequences of paracingulin in modulating junction assembly and targeting to the actin cytoskeleton

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