Membrane palmitoylated proteins regulate trafficking and processing of nectins☆
Introduction
Formation of cell–cell junctions, such as tight junctions in epithelial cells or puncta adherens junctions at synapses, requires the assembly of a highly ordered protein complex containing receptors, signaling molecules, and scaffolding proteins. These assemblies play roles in regulating various cellular processes that govern cellular functions and tissue organization. One of the major key players for the assembly and maintenance of cell–cell junctions is cell adhesion molecules (CAMs) (Dalva et al., 2007, McAllister, 2007, Niessen, 2007).
Recently, nectin, a member of immunoglobulin (Ig)-like CAMs, has been described as a major organizer for the various cell junctions (Takai et al., 2008a, Takai and Nakanishi, 2003). The nectin family is composed of 4 members, nectin-1, -2, -3 and -4, and two or three splicing variants have been found for each isoform (Takai et al., 2003, Takai and Nakanishi, 2003). Nectins have one extracellular region composed of three Ig-like loops, one transmembrane domain, and one cytoplasmic tail (Lopez et al., 1995). Nectin-1, nectin-2 and nectin-3 are expressed ubiquitously in various cell types including epithelia, fibroblasts and neurons, whereas nectin-4 is mainly expressed in the human placenta (Takai and Nakanishi, 2003). Nectins connect to the actin-based cytoskeleton through interactions with afadin, which is an F-actin binding protein (Mandai et al., 1997, Takai and Nakanishi, 2003). Most of the nectin family has a class type II PDZ binding motif. Through this motif, nectins interact with the PDZ domain of afadin (Mandai et al., 1997, Miyahara et al., 2000, Reymond et al., 2001, Satoh-Horikawa et al., 2000). The nectin–afadin system initiates adherens junction formation in epithelial and fibroblast cells before E- or N-cadherin forms cell–cell adhesions (Hoshino et al., 2005, Sato et al., 2006). Nectins further promote the formation of tight junctions by recruiting other molecules such as JAM-A, claudin, and occludin (Fukuhara et al., 2002, Kuramitsu et al., 2008). Nectin-1 and nectin-3 also interact with Par3 and localize together at adherens junctions of the neuroepithelial cells of the embryonic telencephalon (Takekuni et al., 2003). This recruitment establishes the polarity of epithelial cells.
To identify the molecular components of nectin-based cell–cell junctions, we searched for cytoplasmic proteins that interact with the intracellular domain of nectin-1α using a yeast two-hybrid system. We identified MPP3 as a new nectin cytoplasmic interacting molecule. MPP3 belongs to the membrane palmitoylated protein family that is composed of seven members (MPP1 to MPP7) (Gosens et al., 2008). MPP family proteins belong to the p55 Stardust family of membrane-associated guanylate kinase (MAGUK) family of proteins. The Drosophila Stardust protein is required for the establishment of cell polarity in the developing Drosophila embryos (Knust et al., 1993, Tepass and Knust, 1993). The MPP3 gene was identified by virtue of its genomic location to human chromosome 17q12-21 adjacent to the BRCA1 tumor suppressor locus (Fukuhara et al., 2003, Sakurai-Yageta et al., 2009). MPP3 localizes to the retinal outer limiting membrane by binding directly to family member MPP5 also known as Protein Associated with Lin Seven 1 (PALS1) (Kantardzhieva et al., 2006). This suggests that MPP3 may play a role in the maintenance of retinal integrity by regulation of cell adhesion between photoreceptors and Müller glial cells. MPP3 increases cell surface expression of the 5-HT2C receptor and prevents its desensitization in cortical neurons (Gavarini et al., 2006). However, the majority of the biological function of MPP3 is still unknown.
In this study, we characterized the interaction between nectin-1α and MPP3 in vivo and in vitro. Nectin-1α recruits MPP3 to cell–cell contact sites and its association with MPP3 significantly increases cell surface expression and ectodomain shedding of nectin-1α, indicating that MPP3 is involved in trafficking and processing of nectin-1α. Then, we further demonstrated that MPP5, another member of the MPP family, also associates with nectins. This association recruits MPP5 to cell–cell contact sites and increases cell surface expression of nectins. These data suggest that wide interactions between nectins and MPP family members may occur in various cell–cell junctions and that these associations regulate trafficking and processing of nectins.
Section snippets
Cell lines, antibodies, and plasmids
COS-7 and HEK 293A cells were purchased from American Type Culture Collection. All cell lines were maintained in DMEM supplemented with 10% fetal bovine serum and antibiotics (Invitrogen, Carlsbad, CA). For biochemical experiments, COS-7 and HEK-293 cells were plated to a final confluence of 80%. For immunofluorescence experiments, COS-7 cells were plated to a final confluence of 40%. Rabbit anti-nectin-1 antibodies were prepared as described (Lim et al., 2008). Rabbit polyclonal anti-MPP3
Nectin-1 interacts with MPP3 in yeast
To identify potential cytoplasmic binding proteins with nectins, the cytoplasmic domain of nectin-1α was used as a bait to screen a mouse brain cDNA library using the CytoTrap yeast two-hybrid system. After screening 1 million cDNA clones, we found five independent clones. The DNA sequencing analyses revealed that two of the five clones contained the Lin2/Lin2 binding domain (L27), PDZ domain, and SH3 domain of mouse MPP3 (Fig. 1A). MPP3 is one of the MAGUK family proteins, consisting of two
Discussion
In this study, we have found that MPP3, one of the human homologues of Drosophila Discs large, is a new binding partner of nectin-1α. Two major forms of MPP3 at 66 and 98 kDa were detected in various tissues examined (Fig. 2E), in conjunction with nectin-1α, suggesting that an association between the two may occur in various tissues. Nectin-1α specifically associated with the high molecular weight form of MPP3 in the rat brain (Fig. 2F), suggesting that post-translational modification may
Acknowledgement
We thank Dr. Katherine Conant for comments on the manuscript.
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2016, Biochemical PharmacologyCitation Excerpt :It has previously been shown that Lin7 family proteins localize to calcium-insensitive nectin-based cell-cell junctions in MDCK cells and other cell types [30]. This localization is due to an indirect interaction between Lin7a and nectin [31] via MPP3 [32,33] which was shown to be recruited to nectin based cell-cell contacts [34]. Since we had identified MPP3 as a likely indirect binding partner of the α2 PDZ ligand (see Fig. 4), we hypothesized that localization of the α2/β1 complex to calcium-insensitive cell-cell contacts in HEK293 cells could be mediated by Lin7a via MPP3 to nectin.
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2016, Seminars in Cell and Developmental BiologyCitation Excerpt :For instance, trafficking and processing of nectins are modulated by their interacting partners such as cytoplasmic binding partners. Membrane palmitoylated proteins (MPPs) are associated with various nectins via PDZ-binding motif [81]. Association with MPP3 enhances the expression of nectin-1α at the cell surface and promotes the ectodomain shedding of nectin-1α, which in turn regulates the processing and trafficking of nectin-1α [81].
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2015, Current Topics in Developmental BiologyThe CRB1 and adherens junction complex proteins in retinal development and maintenance
2014, Progress in Retinal and Eye ResearchCitation Excerpt :In the retina, MPP3 is localized at the subapical region adjacent to AJs and at the outer plexiform layer where MPP3 interacts with PALS1/MPP5 and DLG1, respectively (Gavarini et al., 2006; Kantardzhieva et al., 2006). Furthermore, in vitro experiments showed that MPP3 can also interact with the AJs proteins Nectin-1 and -3 (Dudak et al., 2011). Ultrastructural localization studies revealed that MPP3 is predominantly localized at the subapical region in apical villi of Müller glial cells (Dudok et al., 2013a, 2013b).
Membrane palmitoylated protein 3 promotes hepatocellular carcinoma cell migration and invasion via up-regulating matrix metalloproteinase 1
2014, Cancer LettersCitation Excerpt :Also, studies implied that MPP3 might participated in the process of human tumorigenesis. MPP3 associates with nectin-1 mediated by its PDZ domain and this association increases cell surface expression and ectodomain shedding of nectin-1 [12]. In addition, the expression of nectin-1on cell surface is up-regulated in highly migratory and invasive carcinoma [13], suggesting there might be a relationship between MPP3 and tumor metastasis.
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Funding for this project was provided by NIH-RO1 AG027233-01.