Crystal structure of afadin PDZ domain–nectin‐3 complex shows the structural plasticity of the ligand‐binding site

Fujiwara, Yoshi; Goda, Nobuhito; Tamashiro, Tomonari; Narita, Hiroko; Satomura, Kimio; Tenno, Takeshi; Nakagawa, Atsushi; Oda, Masayuki; Suzuki, Mamoru; Sakisaka, Toshiaki; Takai, Yoshimi; Hiroaki, Hidekazu

doi:10.1002/pro.2628

Cited by 14 publications

(21 citation statements)

References 51 publications

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“…Nectin proteins consist of a three extracellular Ig domains, a transmembrane fragment, and multiple intracellular C-terminal domains with distinct and independent functions [ 11 , 30 , 62 – 64 ]. Most nectins, including nectin-3, have a conserved motif of four amino acids at their cytoplasmic tail that binds the PDZ domain of afadin [ 11 , 27 , 30 , 40 ]. Nectin-3/afadin binding is required for the interaction of nectin-3 with the actin cytoskeleton and the organization of PAJs in cooperation with N-cadherin (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We next tested whether the decreased spine densities observed with nectin-3 overexpression required an interaction between nectin-3 and afadin. For this experiment, we used in utero electroporation to overexpress a nectin-3 protein lacking the four C-terminal amino acids required to bind afadin (Nec3 Δafadin ) [ 11 , 27 , 30 , 40 ]. Developing L2/3 neurons were electroporated with Nec3 Δafadin (plus Cre and Cre-dependent tdTomato plasmids, Fig.…”

Section: Resultsmentioning

confidence: 99%

“…This expression pattern suggests a potential role for nectin-3 in the development of L2/3-specific visual response properties after eye opening [ 25 ]. We assessed the dendritic spine densities of apical and basal dendrites on L2/3 visual cortical neurons at three developmental time points (P14, P21, and P35) after in utero electroporation of plasmid constructs expressing nectin-3 shRNA, full-length nectin-3, or nectin-3 lacking the four amino acid afadin-binding domain (Nec3 Δafadin ) [ 30 , 40 , 41 ]. Nectin-3 knockdown by shRNA beginning at embryonic day (E)15.5 or, using CaMKII-Cre transgenic mice [ 42 ], at ~P19, increased dendritic spine densities at P21 or P35, respectively.…”

Section: Introductionmentioning

confidence: 99%

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Precise levels of nectin-3 are required for proper synapse formation in postnatal visual cortex

et al. 2020

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Background Developing cortical neurons express a tightly choreographed sequence of cytoskeletal and transmembrane proteins to form and strengthen specific synaptic connections during circuit formation. Nectin-3 is a cell-adhesion molecule with previously described roles in synapse formation and maintenance. This protein and its binding partner, nectin-1, are selectively expressed in upper-layer neurons of mouse visual cortex, but their role in the development of cortical circuits is unknown. Methods Here we block nectin-3 expression (via shRNA) or overexpress nectin-3 in developing layer 2/3 visual cortical neurons using in utero electroporation. We then assay dendritic spine densities at three developmental time points: eye opening (postnatal day (P)14), one week following eye opening after a period of heightened synaptogenesis (P21), and at the close of the critical period for ocular dominance plasticity (P35). Results Knockdown of nectin-3 beginning at E15.5 or ~ P19 increased dendritic spine densities at P21 or P35, respectively. Conversely, overexpressing full length nectin-3 at E15.5 decreased dendritic spine densities when all ages were considered together. The effects of nectin-3 knockdown and overexpression on dendritic spine densities were most significant on proximal secondary apical dendrites. Interestingly, an even greater decrease in dendritic spine densities, particularly on basal dendrites at P21, was observed when we overexpressed nectin-3 lacking its afadin binding domain. Conclusion These data collectively suggest that the proper levels and functioning of nectin-3 facilitate normal synapse formation after eye opening on apical and basal dendrites in layer 2/3 of visual cortex.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Precise levels of nectin-3 are required for proper synapse formation in postnatal visual cortex

et al. 2020

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“…These intracellular proteins participate in cellular signal transduction and thereby regulate a series of cell behaviors [ 35 ]. In present study, afadin, which contains a PDZ domain ligand [ 36 – 39 ], was identified as a potential target gene of CLDN2 protein.…”

Section: Discussionmentioning

confidence: 99%

CLDN2 inhibits the metastasis of osteosarcoma cells via down-regulating the afadin/ERK signaling pathway

Zhang

Wang

et al. 2018

Cancer Cell Int

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BackgroundIn an earlier study, we investigated the expression of tight junction protein claudins (CLDNs) in human osteosarcoma (OS) cells, and the CLDN2 was found to be down-regulated in primary tumor cells compared with normal osteoblast cells. Here, we sought to explore the effects of CLDN2 on the malignant phenotype of OS and the underlying molecular mechanisms.MethodsThe expression patterns of CLDN2 and afadin in OS tissues and histologically non-neoplastic bone tissues were explored via immunohistochemistry and western blotting. CLDN2 expression levels in an OS cell line stably expressing CLDN2 and an osteoblast cell line with a CLDN2 knockout were confirmed by western blotting and immunofluorescence staining. The malignant phenotype of OS cells and osteoblast cells in vitro was assessed using a cell counting kit-8 assay, transwell assay and wound-healing experiment. Western blotting was utilized to detect the activation state of Ras/Raf/MEK/ERK pathway. Moreover, an RNA interference method were used to silence afadin in CLDN2-expressing OS cells.ResultsOur research group found that CLDN2 and afadin was underexpressed in OS tissues, and the overexpression of CLDN2 significantly inhibited the migration abilities of OS cells. Genetic silencing of afadin in CLDN2-overexpressing OS cells promoted U2OS cell motility and activation of the Ras/Raf/MEK/ERK pathway.ConclusionsIn this study, we confirmed that CLDN2 expression significantly inhibited the malignant phenotype of OS cells in vitro. Inhibition of the ERK pathway by afadin may be one of the mechanisms by which CLDN2 blocks the metastasis phenotype of OS cells.

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“…We, and others, have demonstrated that CPAF is capable of degrading host cytoskeletal proteins vimentin and keratin 18 and nuclear envelope protein LAP1 [ 13 – 15 , 69 ]. Interestingly, PDZ domain-mediated association of proteins with cytoskeletal machinery is plausible and has been observed for several proteins, including PDZ-GEF1, afadin, Dlg1, the PDZ-LIM family protein RIL and Lin-7 [ 70 – 74 ]. Because CPAF partially proteolyzes the vimentin cage surrounding the parasitophorous inclusion, we suggest that CPAF 106-212 may facilitate localization of the enzyme to interact with vimentin, vimentin binding proteins, or associated cytoskeletal filaments.…”

Section: Discussionmentioning

confidence: 99%

The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins

et al. 2016

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The need for more effective anti-chlamydial therapeutics has sparked research efforts geared toward further understanding chlamydial pathogenesis mechanisms. Recent studies have implicated the secreted chlamydial serine protease, chlamydial protease-like activity factor (CPAF) as potentially important for chlamydial pathogenesis. By mechanisms that remain to be elucidated, CPAF is directed to a discrete group of substrates, which are subsequently cleaved or degraded. While inspecting the previously solved CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal PSD95 Dlg ZO-1 (PDZ) domain spanning residues 106–212 (CPAF106-212). This PDZ domain is unique in that it bears minimal sequence similarity to canonical PDZ-forming sequences and displays little sequence and structural similarity to known chlamydial PDZ domains. We show that the CPAF106-212 sequence is homologous to PDZ domains of human tight junction proteins.

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Crystal structure of afadin PDZ domain–nectin‐3 complex shows the structural plasticity of the ligand‐binding site

Cited by 14 publications

References 51 publications

Precise levels of nectin-3 are required for proper synapse formation in postnatal visual cortex

Precise levels of nectin-3 are required for proper synapse formation in postnatal visual cortex

CLDN2 inhibits the metastasis of osteosarcoma cells via down-regulating the afadin/ERK signaling pathway

The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins

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