Membrane palmitoylated proteins regulate trafficking and processing of nectins

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Abstract

Nectins are cell–cell adhesion molecules involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell–cell adhesion sites. To have a better understanding of the roles of nectins in the formation of cell–cell junctions, we searched for new cytoplasmic binding partners for nectin. We report that nectin-1α associates with membrane palmitoylated protein 3 (MPP3), one of the human homologues of a Drosophila tumor suppressor gene, Disc large. Two major forms of MPP3 at 66 and 98 kDa were detected, in conjunction with nectin-1α, suggesting that an association between the two may occur in various cell types. Nectin-1α recruits MPP3 to cell–cell contact sites, mediated by a PDZ-binding motif at the carboxyl terminus of nectin-1α. Association with MPP3 increases cell surface expression of nectin-1α and enhances nectin-1α ectodomain shedding, indicating that MPP3 regulates trafficking and processing of nectin-1α. Further study showed that MPP3 interacts with nectin-3α, but not with nectin-2α, showing that the association of nectins with MPP3 is isoform-specific. MPP5, another MPP family member, interacts with nectins with varying affinity and facilitates surface expression of nectin-1α, nectin-2α, and nectin-3α. These data suggest that wide interactions between nectins and MPP family members may occur in various cell–cell junctions and that these associations may 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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    Funding for this project was provided by NIH-RO1 AG027233-01.

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