Coxsackie adenovirus receptor (CAR) regulates integrin function through activation of p44/42 MAPK

Abstract

The coxsackie B virus and adenovirus receptor (CAR) is an attachment receptor for Adenovirus serotype 5 (Ad5) and in many cell types forms homodimers with neighbouring cells as part of a cell adhesion complex. CAR co-operates with cell surface integrin receptors to enable efficient viral entry, but little is known about the mechanism of crosstalk between these two receptor types. Here we show that overexpression of CAR in human epithelial cells leads to increased basal activation of p44/42 MAPK and this is required for efficient Ad5 infection. We demonstrate that CAR forms homodimers in cis and that this dimerisation is enhanced in the presence of Ad5 in a phospho-p44/42-dependent manner. CAR-induced p44/42 activation also leads to increased activation of β1 and β3 integrins. Analysis of CAR mutants demonstrates that the cyto domain of CAR is required for CAR-induced p44/42 activation, integrin activation and localisation to cell junctions. This data for the first time demonstrates that signalling downstream of CAR can have a dual effect on integrins and CAR itself in order to promote efficient viral binding to cell membranes.

Introduction

Adenovirus serotype 5 (Ad5) is one of the most widely used viral transduction systems for high-level delivery of therapeutic gene products. Ad5 is a non-enveloped DNA virus and consists of an inner core and an outer capsid. The virus capsid contains 12 vertices that are composed of the penton base and fibre proteins. The fibre and RGD (Arg-Gly-Asp) motif containing penton base proteins in the capsid are important in Ad5 attachment to cells via CAR and integrins respectively [1]. Ad5 infection is achieved through primary attachment to CAR followed by integrin-dependent clathrin and dynamin-mediated endocytosis [1], [2]. Integrins αvβ3, αvβ5, α5β1 and αvβ1 have all been shown to mediate Ad5 internalisation [3], [4]. Of these, αvβ3 has received considerable attention as a key integrin involved in virus uptake in cancer-derived cells [5], [6].

CAR is a 46 kDa transmembrane receptor and a member of the immunoglobulin (Ig) superfamily. The extracellular domain contains two Ig-like regions (D1 and D2) and D1 has been shown to form a complex directly to the Ad knob [7].

Much is now known about the interaction of Ad with CAR and yet surprisingly little is known about the biological function of CAR. CAR is a component of the tight junction where it colocalises and precipitates with the membrane-anchored scaffold plaque tight junction protein zonula occludens-1 (ZO-1) [8]. The C-terminal cytoplasmic domain of CAR is highly conserved between species, contains putative phosphorylation sites and a membrane distal PDZ domain. Studies have shown that the amount of CAR expressed on cells can regulate levels of Ad infection. It is thought that CAR is only involved in initial Ad attachment as deletion of the cytoplasmic and transmembrane domains does not alter Ad infection [9], [10]. CAR can colocalise with integrins αvβ3 and αvβ5 [11] and has been found to reside in a novel lipid raft domain. This has been proposed as a mechanism by which CAR can interact with integrins and allow Ad internalisation [12]. Alternatively, the integrin may respond to a signal from CAR as opposed to a direct binding event, or from the Ad particle itself.

Here we show that high levels of CAR expression in human epithelial cells lead to high basal activation of p44/42 MAPK. This enhanced signalling has two outcomes; control of self-dimerisation and clustering of CAR at the membrane in response to Ad5 binding as well as a secondary effect upon cell:matrix adhesion, integrin activation and subcellular localisation. We postulate that this represents a mechanism by which CAR can regulate a feedback loop to promote efficient Ad5 membrane docking to CAR itself whilst also controlling the secondary Ad5 integrin receptors to promote efficient viral internalisation.