PLEKHA7 Recruits PDZD11 to Adherens Junctions to Stabilize Nectins

Abstract

PLEKHA7 is a junctional protein implicated in stabilization of the cadherin protein complex, hypertension, cardiac contractility, glaucoma, microRNA processing, and susceptibility to bacterial toxins. To gain insight into the molecular basis for the functions of PLEKHA7, we looked for new PLEKHA7 interactors. Here, we report the identification of PDZ domain-containing protein 11 (PDZD11) as a new interactor of PLEKHA7 by yeast two-hybrid screening and by mass spectrometry analysis of PLEKHA7 immunoprecipitates. We show that PDZD11 (17 kDa) is expressed in epithelial and endothelial cells, where it forms a complex with PLEKHA7, as determined by co-immunoprecipitation analysis. The N-terminal Trp-Trp (WW) domain of PLEKHA7 interacts directly with the N-terminal 44 amino acids of PDZD11, as shown by GST-pulldown assays. Immunofluorescence analysis shows that PDZD11 is localized at adherens junctions in a PLEKHA7-dependent manner, because its junctional localization is abolished by knock-out of PLEKHA7, and is rescued by re-expression of exogenous PLEKHA7. The junctional recruitment of nectin-1 and nectin-3 and their protein levels are decreased via proteasome-mediated degradation in epithelial cells where either PDZD11 or PLEKHA7 have been knocked-out. PDZD11 forms a complex with nectin-1 and nectin-3, and its PDZ domain interacts directly with the PDZ-binding motif of nectin-1. PDZD11 is required for the efficient assembly of apical junctions of epithelial cells at early time points in the calcium-switch model. These results show that the PLEKHA7-PDZD11 complex stabilizes nectins to promote efficient early junction assembly and uncover a new molecular mechanism through which PLEKHA7 recruits PDZ-binding membrane proteins to epithelial adherens junctions.

Cell‐specific diversity in the expression and organization of cytoplasmic plaque proteins of apical junctions

“…). The known interacting partners of PLEKHA7 (i.e., PDZD11 and afadin) were detected in PLEKHA7 IPs from both meEC and mCCD cells (Fig. A), in similar amounts, as determined by quantitative analysis of blots (Fig.…”

“…Next, we examined the expression of the cytoplasmic proteins associated with cadherins: PLEKHA7 and its interactors afadin, p120‐ctn, PDZD11, and α‐catenin, the latter being indirectly associated with cadherin through β‐catenin . PLEKHA7 expression levels were highest in mCCD, A427, and A549 cells, and PLEKHA7 was detected in all other cell types, except for H5V cells (Fig.…”

“…To confirm PLA results using a biochemical approach, we immunoprecipitated either PLEKHA7 or its interactor PDZD11 from lysates of either endothelium‐derived (meEC) or epithelial (mCCD) cells, and we examined the presence of either afadin, ZO‐1, or cingulin in IPs by IB analysis (Fig. ).…”

“…The following primary antibodies were used for IB and IF: rabbit anti‐cingulin C532 (1:5000 IB, 1:10000 IF), mouse anti‐cingulin 22BD5A1 (1:30 IB), rabbit anti‐paracingulin n.821 (1:10000 IB, 1:100 IF), mouse anti‐paracingulin (E‐6) (1:1000 IB, Santa Cruz, sc‐377525), mouse anti‐ZO‐1 (1:1000 IB, 1:5000 IF, Invitrogen, 33–9100), rabbit anti‐ZO‐2 (H‐110) (1:1000 IB, Santa Cruz, sc‐11448), rabbit anti‐ZO‐3 (1:2000 IB, Invitrogen, 36–4100), mouse anti‐occludin (1:1000 IB, 1:100 IF, Invitrogen, 33–1500), rabbit anti‐occludin (1:1000 IB, 1:100 IF, Invitrogen, 71–1500), rabbit anti‐PLEKHA7 R30388 (1:2000 IB, 1:500 IF), guinea pig anti‐PLEKHA7 GP2737 (1:500 IF), rabbit anti‐afadin (1:8000 IB, 1:100 IF, Sigma, A0224), rabbit anti‐PDZD11 (1:1000 IB, 29958), mouse anti‐p120‐catenin (1:2000 IB, 1:250 IF, 15D2, a kind gift of Prof. A. Reynolds, Vanderbilt University Nashville, TN,) rabbit anti‐α‐catenin (1:8000 IB, 1:500 IF, Sigma, C2081), mouse anti‐E‐cadherin (1:8000 IB, 1:2000 IF, BD610181, which crossreacts with P‐cadherin (http://www.bdbiosciences.com/ds/pm/tds/610181.pdf)), goat anti‐VE‐cadherin (C‐19) (1:1000 IB, 1:50 IF, Santa Cruz, sc‐6458), rabbit anti‐pan‐cadherin (1:1000 IB, 1:100 IF, Sigma, C3678), and mouse anti‐β‐tubulin (1:4000 IB, Invitrogen, 32–2600). The specificity of primary antibodies against CGN, CGNL1, PLEKHA7, and PDZD11 was validated in our laboratory using depleted or knockout (KO) cells.…”

“… and ). The cytoplasmic regions of nectins interact with afadin, which directly interacts with actin filaments and with PDZD11, which is recruited to the ZA by PLEKHA7 . Classical cadherins exist in different isoforms; for example, E‐cadherin accumulates at AJs of epithelial cells, whereas VE‐cadherin accumulates at AJs of endothelial cells .…”