A Role for ZO-1 and PLEKHA7 in Recruiting Paracingulin to Tight and Adherens Junctions of Epithelial Cells

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 cadherinbased cell-cell junctions in Rat1 fibroblasts requires the ZIMcontaining 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. Tight junctions (TJ)2 and adherens junctions (AJ) are key elements of the apical junctional complex of vertebrate epithelial cells and are of fundamental importance in the morphogenesis and homeostasis of epithelial tissues. TJ and AJ carry out several functions: canonical roles in cell-cell adhesion and tissue sorting (AJ), cell polarization and permeability barrier functions (TJ), and signaling functions through pathways that control the organization of the cytoskeleton and the activity of transcription factors (for review, see Refs.

Binding between the Junctional Proteins Afadin and PLEKHA7 and Implication in the Formation of Adherens Junction in Epithelial Cells

“…4). In addition, another PLEKHA7-binding protein paracingulin is localized at both AJ and TJ (20). These three PLEKHA7-binding proteins all are localized at AJ, although p120 ctn and paracingulin are distributed to other sites than AJ.…”

“…However, paracingulin is localized not only at AJ but also at TJ (21). It was proposed that paracingulin is localized at AJ and TJ by binding to PLEKHA7 and ZO-1, respectively (20). Thus, it still remains unknown how PLEKHA7 is localized strictly at AJ in epithelial cells.…”

“…In contrast, PLEKHA7 is strictly localized at AJ like the nectinafadin system in Caco2 cells (19). It was more recently shown that PLEKHA7 binds to paracingulin, which is known to interact with ZO-1, and is localized at AJ in Madin-Darby canine kidney (MDCK) cells (20). However, paracingulin is localized not only at AJ but also at TJ (21).…”

PLEKHA7 Recruits PDZD11 to Adherens Junctions to Stabilize Nectins

“…GST fusions of smaller fragments of PLEKHA7 (WW, 1-162), WWPH(1-284), PH(120 -300), and proline-rich ϩ coiled-coil region (PCC, 500 -844) were generated by PCR and subcloned into pGEX4T1. Other GST fusions of PLEKHA7 were described previously (7,8). The internal WW and WF domains within the larger WW region (1-162) were subsequently identified and subcloned into pGEX4T1 to make three GST fusion constructs: W1 (WW, 1-53), W2 (WF, , and W1 ϩ 2 (WW-WF, 1-98).…”

“…PLEKHA7 is a recently discovered cytoplasmic component of AJ, which was identified through its interaction with the AJ protein p120-catenin (6) and with the TJ/AJ protein paracingulin (CGNL1) (7)(8)(9). PLEKHA7 is specifically associated with the zonula adhaerens (ZA) (7), and it interacts with the microtubule-interacting protein nezha (CAMSAP3) to provide a molecular linkage between the cadherin-associated protein complex and the microtubule cytoskeleton (6).…”