Afadin- and α-actinin-binding protein ADIP directly binds β′-COP, a subunit of the coatomer complex

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Abstract

Afadin DIL domain-interacting protein (ADIP) is a novel protein that binds both afadin and α-actinin and localizes at adherens junctions, which are formed by nectins and cadherins, cell–cell adhesion molecules. Afadin is an actin filament (F-actin)-binding protein which connects nectins to the actin cytoskeleton. α-Actinin is another F-actin-binding protein that is indirectly associated with cadherins through the catenin complex. ADIP is at least partly involved in the physical association of nectins and cadherins. We show here that ADIP furthermore binds β′-COP, a subunit of the coatomer complex. ADIP co-localizes with β′-COP at the Golgi complex in Madin Darby canine kidney and normal rat kidney cells. These results suggest that ADIP is involved in vesicle trafficking from the Golgi to the endoplasmic reticulum and through the Golgi complex by interacting with the coatomer complex.

Section snippets

Materials and methods

Yeast two-hybrid screening. One bait vector, pGBD-mADIP-M (amino acids (aa) 152–436), was constructed by subcloning the insert encoding aa residue of ADIP into pGBD-C1 [8]. Yeast two-hybrid library constructed from the rat lung cDNAs was purchased from Clontech. The two-hybrid screening was performed as described [8]. Standard procedures for yeast manipulations were done as described [9].

Construction of expression vectors. The mammalian expression vectors, pCMV-Flag and pCMV-HA, were designed

Identification of β′-COP as an ADIP-binding protein

To identify an ADIP-binding protein, we performed the yeast two-hybrid screening using the region of ADIP (aa 152–436) containing the three coiled-coil domains as a bait (Fig. 1A). We screened 2 × 105 clones of a rat lung library and obtained 10 positive clones. Three rat clones encoded the carboxyl-terminal portion (aa 370–905) of rat β′-COP (AF002705; GenBank/EMBL/DDBJ) (Fig. 1B). β′-COP contains six internal repeats of the WD40 motif, but the carboxyl-terminal portion (aa 370–905) is not

Discussion

We have shown here that ADIP, an afadin- and α-actinin-binding protein, localizes at the Golgi complex and directly binds β′-COP, a subunit of the coatomer complex. β′-COP is a subunit of the coatomer complex and this complex consists of α-, β-, β′-, γ-, δ-, ε-, and ζ-COPs and is involved in vesicle trafficking from the Golgi to the ER and through the Golgi complex [6], [7]. However, it remains unknown whether the interaction of ADIP with β′-COP is involved in this vesicle trafficking. It has

Acknowledgment

We thank Dr. W. Birchmeier (Max-Delbruck-Center for Molecular Medicine, Berlin, Germany) for providing us with MDCK cells. This investigation was supported by Grants-in-Aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, Culture, and Technology, Japan (2002, 2003).

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    Abbreviations: aa, amino acids; ADIP, afadin DIL domain-interacting protein; AJs, adherens junctions; ER, endoplasmic reticulum; F-actin, actin filament; GST, glutathione S-transferase; HA, hemagglutinin; MDCK cell, Madin Darby canine kidney cell; MBP, maltose-binding protein; NRK cell, normal rat kidney cell; pAb, polyclonal antibody; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.

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    Present address: Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, 1-1, Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan.

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