The Coxsackie and Adenovirus Receptor Binds Microtubules and Plays a Role in Cell Migration

Abstract

The Coxsackie and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, inhibits cell growth of a variety of tumors. The cytoplasmic domain of CAR has been implicated in decreased invasion and intracerebral growth of human U87 glioma cells. Using affinity binding, we identified tubulin as an interaction partner for the cytoplasmic domain of CAR. The interaction was specific; CAR and tubulin co-immunoprecipitated in cells expressing endogenous CAR and partially co-localized in situ. The binding of CAR to tubulin heterodimers and to microtubules was direct, with dissociation constants of ∼1 μm for tubulin and ∼32 nm for in vitro assembled microtubules. Whereas CAR-expressing U87 glioma cells had decreased migration in a chemotactic assay in Boyden chambers as compared with control cells, an effect that depended on the presence of the cytoplasmic domain of CAR, the difference was abrogated at low, non-cytotoxic doses of the taxane paclitaxel, a microtubule-stabilizing agent. These results indicate that CAR may affect cell migration through its interaction with microtubules. The Coxsackie and adenovirus receptor (CAR), a cell adhesion molecule of the immunoglobulin superfamily, inhibits cell growth of a variety of tumors. The cytoplasmic domain of CAR has been implicated in decreased invasion and intracerebral growth of human U87 glioma cells. Using affinity binding, we identified tubulin as an interaction partner for the cytoplasmic domain of CAR. The interaction was specific; CAR and tubulin co-immunoprecipitated in cells expressing endogenous CAR and partially co-localized in situ. The binding of CAR to tubulin heterodimers and to microtubules was direct, with dissociation constants of ∼1 μm for tubulin and ∼32 nm for in vitro assembled microtubules. Whereas CAR-expressing U87 glioma cells had decreased migration in a chemotactic assay in Boyden chambers as compared with control cells, an effect that depended on the presence of the cytoplasmic domain of CAR, the difference was abrogated at low, non-cytotoxic doses of the taxane paclitaxel, a microtubule-stabilizing agent. These results indicate that CAR may affect cell migration through its interaction with microtubules. CAR 5The abbreviations used are: CAR, Coxsackie and adenovirus receptor; AdVBFP, adenovirus expressing blue fluorescent protein; AdVAS, antisense full-length CAR; PBS, phosphate-buffered saline; GST, glutathione S-transferase; BSA, bovine serum albumin; MT, microtubule; MAP2, microtubule-associated protein 2; PIPES, 1,4-piperazinediethanesulfonic acid. (Coxsackie and adenovirus receptor) is a 46-kDa plasma membrane glycoprotein (1Bergelson J.M. Cunningham J.A. Droguett G. Kurt-Jones E.A. Krithivas A. Hong J.S. Horwitz M.S. Crowell R.L. Finberg R.W. Science. 1997; 275: 1320-1323Crossref PubMed Scopus (2657) Google Scholar, 2Tomko R.P. Xu R. Philipson L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3352-3356Crossref PubMed Scopus (1048) Google Scholar), which constitutes the primary site of attachment for all serotypes of Coxsackie B virus and for many adenovirus serotypes (3Roelvink P.W. Lizonova A. Lee J.G. Li Y. Bergelson J.M. Finberg R.W. Brough D.E. Kovesdi I. Wickham T.J. J. Virol. 1998; 72: 7909-7915Crossref PubMed Google Scholar, 4Nemerow G.R. Virology. 2000; 274: 1-4Crossref PubMed Scopus (162) Google Scholar). CAR belongs to a newly recognized subclass of the immunoglobulin superfamily of transmembrane proteins termed the CTX family (5Chretien I. Marcuz A. Courtet M. Katevuo K. Vainio O. Heath J.K. White S.J. Du P.L. Eur. J. Immunol. 1998; 28: 4094-4104Crossref PubMed Google Scholar). Members of the CTX subclass of immunoglobulin superfamily proteins are homologous type I transmembrane proteins with an extracellular moiety consisting of one variable (V-type) and one constant (C2-type) immunoglobulin (Ig) domain, a single transmembrane domain, and a cytoplasmic tail. The founding member of the CTX subfamily (cortical thymocy