Tetraspanins: integrating cell surface receptors to functional microdomains in homeostasis and disease

The translation of extracellular signaling cues into intracellular signaling cascades is frequently mediated by the assembly of signaling complexes at the plasma membrane. Different mechanisms have evolved how these signaling complexes propagate the signals into the interior of the recipient cell. Signaling by G-protein-coupled receptors (GPCRs) is mediated by conformational changes induced by ligand binding resulting in the dissociation of heterotrimeric G-proteins and activation of signaling cascades through the generation of second messengers [1]. Signaling through receptor tyrosine kinases (RTKs) generally involves receptor dimerization as a consequence of ligand binding, which results in a release of the intracellular autoinhibited kinase domain into the active configuration [2]. The consequence of trans-phosphorylation is strongly increased kinase activity, phosphorylation of multiple tyrosine residues, recruitment of adaptor proteins, and assembly of signaling complexes [2].

Signaling events are frequently restricted to specific subcellular sites. This site-specific localization of signaling events can be mediated by physically linking the signaling molecules to cell adhesion receptors present at specific locations, such as sites of cell–matrix adhesion like focal adhesions (FA) [3], or sites of cell–cell adhesion like adherens junctions (AJ) [4], tight junctions (TJ) [5], or synapses [6]. One mechanism underlying the assembly of larger signaling complexes at these sites is the use of scaffolding proteins which directly interact with cell adhesion receptors. These scaffolding proteins contain multiple protein–protein interaction domains such as PDZ domains, SH3 domains, or FERM domains [7], allowing them to recruit cytoplasmic signaling proteins at specific sites for functional interactions. One additional mechanism is based on lateral associations of cell adhesion receptors with other integral membrane proteins through linker proteins that are also embedded in the plasma membrane but can interact with several other integral membrane proteins simultaneously. By generating microdomains in the membrane, these lateral associations can promote the assembly of higher order protein complexes.

Tetraspanins (Tspans, also called transmembrane 4 superfamily (TM4SF) proteins), a family of integral membrane proteins consisting of 33 members in humans [8, 9], have emerged as bona fide organizers of microdomains in the plasma membrane [10‐15]. Tspans contain four α-helical transmembrane (TM) regions (TM1–TM4), two extracellular (EC) domains (EC1 and EC2), and three cytoplasmic (CP) regions, (N-terminus, C-terminus and a short loop that connects TM2 and TM3) [10]. These topological domains contribute differently to the structure of tetraspanins and to their ability to undergo intramolecular and intermolecular interactions [15]. The most distinctive feature that qualifies Tspans to organize larger protein complexes in the membrane is their ability to use several of their topological regions of the protein, i.e., the EC domains, the TM domains and the CP regions for interactions with other proteins [16, 17]. Of the extracellular domains, EC2 is predominantly involved in heterophilic interactions with other proteins and mainly responsible for the specificities underling these interactions [18]. In addition, the EC2 domain can also mediate tetraspanin dimerization [19]. The TM domains undergo intramolecular interactions that support the functional protein conformation, but they can also mediate homophilic and heterophilic Tspan–Tspan interactions [15, 16, 20, 21]. The CP regions of some Tspan proteins have been found to interact with cytosolic signaling or scaffolding proteins. These include conventional PKCs, which bind to the N-terminal CP region of CD9 [22], small GTPases like Rac1 which interact with the C-terminal CP region of CD81 [23], and PDZ domain-containing scaffolding proteins like Syntenin-1 or Pick-1, which bind to PDZ domain-binding motifs at the C-termini of CD63 or Tspan7, respectively [24, 25]. The multiple possibilities to interact with other proteins enables Tspans to assemble microdomains or clusters. Tetraspanin clusters cover an area of approximately 100–400 nm² [12, 15, 26, 27]. More recent studies using superresolution microscopy indicate that Tspans form nanoclusters composed of a limited number (less than 10) of selected Tspans with only minor overlaps with other Tspan nanoclusters [15]. The assembly of protein complexes to so-called Tspan-enriched microdomains (TEMs) in the plasma membrane is most liklely the principal biological function of Tspans. In this review article, we will present some examples to illustrate the molecular mechanisms through which Tspans exert their function as organizers of signaling complexes during homeostasis and disease with a particular emphasis on their function in connecting immunoglobulin superfamily (IgSF) proteins to other cell surface receptors.

The regulation of EWI protein interactions with other integral membrane proteins by tetraspanins can be mutual and even occur in a negative manner. As opposed to the recruitment of EWI-2 to α3β1 integrin by CD9 and CD81 to allow functional interaction between EWI-2 and the integrin, the recruitment of CD9 and CD81 to TGFβ receptors is prevented by EWI-2 to limit TGFβ signaling [56].

TGFβ signaling plays a central role during epithelial-to-mesenchymal transition (EMT), a process that is critical during development but when inadvertently reactivated in the adult organism contributes to tumor progression by facilitating invasion and metastasis [57]. TGFβ signals by inducing hetero-dimerization of two receptors, the Ser/Thr kinases TGFβ receptor I (TβRI) and TβRII, resulting in the activation of SMAD proteins, a family of cytoplasmic proteins that form transcriptional complexes which interact with other transcription factors to regulate a plethora of genes [58]. In melanoma cells, TGFβ signaling contributes to the development of an EMT-like phenotype and to mestasasis [59]. Intriguingly, TGFβ signaling has different outcomes in melanocytes versus melanoma cells. It has cytostatic effects in melanocytes but promotes proliferation and survival in melanoma cells [60].

A recent study indicates that the tetraspanin partner proteins EWI-2 negatively regulates TGFβ signaling, and that EWI-2 regulates TGFβ signaling activity through CD9 and CD81 [56]. In early stage melanoma cells, when TGFβ signaling is low, EWI-2 is strongly expressed and interacts with CD9 and CD81, thereby sequestering CD9 and CD81 from the TGFβ receptor complex. However, in later stage melanoma cells when TGFβ signaling is high, EWI-2 is downregulated, which allows its two tetraspanin partners CD9 and CD81 to engage with TGFβ receptors and stabilize a signaling active TGFβ receptor complex resulting in increased Smad signaling [56] (Fig. 4). TGFβ receptor signaling thus provides a scenario in which tetraspanins are sequestered from a signaling receptor in the plasma membrane by interacting with a tetraspanin binding partner.

The B cell receptor (BCR) mediates the antigen-specific activation of B-lymphocytes leading to the production of antibodies. The BCR consists of membrane-bound immunoglobulins (Ig) which bind the antigens, and of the non-covalently bound co-receptors CD79A and CD79B (Igα and Igβ, respectively) which form a heterodimer that initiates intracellular signaling [61]. The intracellular signaling is initiated by phosphorylation of ITAM motifs present in the cytoplasmic domains of CD79A and CD79B by Src family kinases (SFK), which leads to an increased association of these kinases with the co-receptors and enhancement of their kinase activity. These initial signals are then propagated by the recruitment of adaptor proteins which recruit additional kinases, adaptors and phospholipases [61].

In the absence of antigens, the BCR is not silent but generates a low strength signal that is required for the survival of mature B cells [62]. Antigen binding amplifies the tonic low strength signal into a strong signal, suggesting the presence of accessory molecules which serve to increase the signaling capacity of the BCR. One of these accessory molecules is CD19, a single-pass transmembrane protein with two Ig-like domains [63]. CD19 is associated with the BCR and enhances BCR signaling, which is most likely based on its ability to serve as a substrate for SFK and at the same time as a SFK scaffold that promotes SFK activity, and in addition on its ability to regulate PI(3)K signaling [64, 65]. Interestingly, CD19 interacts with tetraspanin CD81 [66, 67]. The interaction with CD81 is necessary for CD19 surface localization [68] but also for the activation of B cells through the BCR [69]. Consistent with this function, the absence of CD81 results in a failure in mounting a humoral immune response [70, 71].

More recent evidence provided a deeper insight into the role of CD81 in regulating BCR signaling through CD19. In resting B cells, IgM- and IgD-based BCRs as well as CD19 are compartimentalized in nanoclusters at the B cell surface. Disrupting the actin cytoskeleton results in increased diffusion of IgM and IgD nanoclusters triggering cytoskeleton-dependent BCR signaling. This observation is interpreted in a way that the BCR is kept immobile in a nanocluster and that its diffusibility is required for efficient BCR signaling. Of note, CD19 is organized in a separate nanocluster, and its diffusibility is not dependent on the actin cytoskeleton but on its association with CD81 [69]. In the absence of CD81, CD19 diffusibility is altered, resulting in impaired B cell activation. CD81 thus serves to immobilize CD19 in a nanocluster separate from the BCR nanoclusters. Antigen binding to the BCR increases BCR diffusion allowing functional cross-talk with CD19-containing CD81-based nanoclusters, which triggers signal amplification [69]. These findings thus highlight a function of tetraspanins in separating co-receptors such as CD19 from primary receptors (i.e., BCRs) to maintain low tonic signaling activity but allow a strong signal amplification by the rapid coalescence of preformed nanoclusters of receptors and their co-receptors (Fig. 5).

One of the most distinctive features of tetraspanins is their ability to associate with other proteins using all of their topological doamins, i.e., their extracellular domains, transmembrane domains and cytoplasmic domains. Together with their ability to undergo homophilic and heterophilic interactions in cis with other tetraspanins, this ability allows tetraspanins to assemble large protein complexes into microdomains. Not surprisingly, tetraspanin-enriched microdomains localized at the plasma membrane are frequently involved in intercellular communication, which requires a rapid assembly of signaling complexes in response to an extracellular signal at a specific subcellular site. Rapid signaling requires close spatial proximity of primary signaling factors, such as growth factor receptors or adhesion receptor-associated signaling molecules, with their regulators. Tetraspanins are ideally suited to assemble protein complexes in which signaling molecules and their regulators are spatially segregated to prevent activation in the absence of agonist but on the other hand are close enough to allow rapid cross-talk in the presence of agonists. Recent observations in other protein-enriched signaling units, i.e., integrin-based adhesion complexes, indicate that signal propagation occurs in the absence of changes in the composition of the adhesion complex, but is regulated by the relay of phosphotyrosine-dependent signals [72]. As outlined in this review article, tetraspanins can regulate proximity as well as spatial separation of functionally interacting proteins and thus provide the structural framework for rapid regulation of signaling processes. The application of high-resolution imaging techniques that allow single protein tracking will be key to a deeper understanding of the molecular mechanism underlying the function of tetraspanins in development, homeostasis and pathophysiology.