Mutation of juxtamembrane cysteines in the tetraspanin CD81 affects palmitoylation and alters interaction with other proteins at the cell surface

Abstract

Palmitoylation of tetraspanins affects protein–protein interactions, suggesting a key role in the assembly of the tetraspanin web. Since palmitoylation occurs on intracellular cysteine residues, we examined whether mutating these residues in the human tetraspanin CD81 would affect the association of CD81 with other surface membrane proteins. Mutation of at least six of the eight juxtamembrane cysteines was required to completely eliminate detectable CD81 palmitoylation, indicating that several sites can be palmitoylated. Interestingly, these mutated proteins exhibited reduced cell surface detection by antibody compared to wild-type CD81, but this was not due to differences in the level of protein expression, trafficking to the cell surface, protein stability, or anti-CD81 antibody binding affinity. Instead, the mutant CD81 proteins appeared to be partially hidden from detection by anti-CD81 antibody, presumably due to altered interactions with other proteins at the cell surface. Associations with the known CD81-interacting proteins CD9 and EWI-2 were also impaired with the mutant CD81 proteins. Taken together, these findings indicate that mutation of juxtamembrane cysteines alters the interaction of CD81 with other proteins, either because of reduced palmitoylation, structural alterations in the mutant proteins, or a combination of both factors, and this affects the CD81 microenvironment on the cell surface.

Introduction

CD81 is a ubiquitously expressed member of the tetraspanin superfamily of proteins. Tetraspanins are defined by four transmembrane domains and by the presence of conserved cyteines in addition to a CCG motif in the large extracellular loop. Their most striking property is the ability to interact with multiple proteins (e.g., receptors, adhesion proteins, Ig superfamily members, and signaling molecules, as well as other tetraspanins). These associations are thought to occur mainly in membrane compartments such as lipid rafts and tetraspanin-enriched microdomains (TEMs), thereby forming a membrane network known as the “tetraspanin web”. Since they regulate the function of their binding partners, tetraspanins play roles in diverse cellular processes including adhesion, proliferation, fusion, and signaling [1], [2]. However, redundancy among the members of the superfamily and the multiplicity of associations make the understanding of their specific molecular mechanism a challenging task.

Research on CD81 has largely focused on its regulatory role in the immune system since its discovery in 1990 as a target of an antiproliferative antibody on lymphoma B-cells [2], [3]. Through its direct association with CD19, CD81 participates in the B-cell coreceptor, a complex of integral membrane proteins (CD19/CD21/CD81) that lowers the threshold of activation of the B-cell receptor in response to opsonized antigens [4], [5]. CD81 is thought to facilitate trafficking of CD19 and stabilize the B-cell coreceptor [6], [7]. Accordingly, B-cells from CD81-null mice express less CD19 on the cell surface and present a weaker antibody response to Th2-inducing antigens [8], [9], [10], [11]. Lack of CD81 also results in a defect in allergen-induced airway hyper-reactivity, a Th2-dependent reaction commonly found in asthma patients [12]. T-cell function is also influenced by CD81 since T-cells isolated from CD81-deficient mice proliferate more rapidly after T-cell receptor cross-linking in comparison with wild-type littermates [9]. Moreover, CD81 interacts with CD4 and CD8, and is able to induce a T-cell costimulatory signal [13], [14], [15]. CD81 has also been found to dynamically concentrate in the immune synapse, a cluster of proteins formed during the close contact between T-cells and antigen-presenting cells essential for T-cell activation [16]. Thus, all these immunological CD81 functions depend on interactions and clustering with other proteins, but how CD81 is able to regulate these associations remains unclear.

It is likely that post-translational modifications have a role in these functions. Palmitoylation is a post-translational process that attaches palmitic acid to proteins via thioester linkages to cysteine residues. Since it is a reversible process, it can be considered an “on–off” switch similar to protein phosphorylation. Addition of fatty acids is thought to provide proteins more hydrophobic stability in cellular membranes, thus influencing their fate during trafficking and partitioning into membrane microdomains [17], [18]. Most tetraspanins possess cysteines in their intracellular domains that may be available for palmitoylation, and all tetraspanins tested so far have been shown to be acylated [19], [20], [21], [22], [23]. Palmitoylation-deficient constructs have been generated for several tetraspanins (CD9, CD81, CD82, and CD151), revealing a role for palmitoylation in protein association, cell motility, morphology, adhesion-dependent signaling, and oxidative stress [20], [21], [22], [23]. In B-cells, coligation of the B-cell receptor with the CD19/CD21/CD81 complex induces CD81 palmitoylation, a necessary event resulting in the localization of this cluster of proteins into lipid rafts and signaling [24]. In contrast, palmitoylation of CD9 and CD151 does not seem to be required for their association with TEMs [19], [20], [21].

In this study, we examined whether mutation of intracellular cysteine residues that are sites of palmitoylation would affect CD81 trafficking and/or interactions with other proteins. To address this issue, we characterized CD81 mutant proteins with substitutions in five, six, seven, or eight cysteines in the cytoplasmic and transmembrane domains. In particular, we assessed CD81 expression at the plasma membrane, palmitoylation levels, association with direct binding partners, and protein stability. Our results highlight the importance of intracellular and transmembrane cysteines for the interaction of CD81 with other proteins at the cell surface.