Abstract
Paracingulin is a 160-kDa protein localized in the cytoplasmic region of epithelial tight and adherens junctions, where it regulates RhoA and Rac1 activities by interacting with guanine nucleotide exchange factors. Here, we investigate the molecular mechanisms that control the recruitment of paracingulin to cell-cell junctions. We show that paracingulin forms a complex with the tight junction protein ZO-1, and the globular head domain of paracingulin interacts directly with ZO-1 through an N-terminal region containing a conserved ZIM (ZO-1-Interaction-Motif) sequence. Recruitment of paracingulin to cadherin-based cell-cell junctions in Rat1 fibroblasts requires the ZIM-containing region, whereas in epithelial cells removal of this region decreases the junctional localization of paracingulin at tight junctions but not at adherens junctions. Depletion of ZO-1, but not ZO-2, reduces paracingulin accumulation at tight junctions. A yeast two-hybrid screen identifies both ZO-1 and the adherens junction protein PLEKHA7 as paracingulin-binding proteins. Paracingulin forms a complex with PLEKHA7 and its interacting partner p120ctn, and the globular head domain of paracingulin interacts directly with a central region of PLEKHA7. Depletion of PLEKHA7 from Madin-Darby canine kidney cells results in the loss of junctional localization of paracingulin and a decrease in its expression. In summary, we characterize ZO-1 and PLEKHA7 as paracingulin-interacting proteins that are involved in its recruitment to epithelial tight and adherens junctions, respectively.
Highlights
Teins of TJ are anchored to the actin cytoskeleton by cytoplasmic scaffolding proteins, among which ZO-1 and ZO-2 play a critical role [11,12,13]
Paracingulin Interacts with ZO-1 through the ZIM-containing Region of the Globular Head Domain and Is Recruited to ZO-1-containing Junctions in a ZIM-dependent Manner—To ask whether paracingulin can interact with ZO-1, we first examined whether paracingulin and ZO-1 form a complex
In this paper we address the molecular mechanisms of recruitment of paracingulin to epithelial junctions, and we characterize PLEKHA7 and ZO-1 as interacting partners of paracingulin, which are implicated in the targeting of paracingulin to epithelial AJ and TJ, respectively (Fig. 5)
Summary
Teins of TJ are anchored to the actin cytoskeleton by cytoplasmic scaffolding proteins, among which ZO-1 and ZO-2 play a critical role [11,12,13]. The paracingulin constructs lacking the ZIM-containing region were not colocalized with ZO-1 (Fig. 1D, ⌬1–110 and ⌬1–209, arrowheads, and magnified insets), albeit they were still detected in a junctional localization.
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