In situ chemical cross-linking on living cells reveals CD9P-1 cis-oligomer at cell surface

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. They associate with each other in multimolecular complexes containing numerous membrane proteins. As a first step towards the study of the supramolecular organization of tetraspanin complexes, we have implemented a proteomic approach based on in situ protein cross-linking on living cells followed by affinity purification of tetraspanin complexes. This allowed observing the presence of high molecular weight protein complexes that were characterized as containing CD9P-1/CD315 using LC-MS/MS. Western blot analyses and the use of different tags demonstrated the presence of CD9P-1 oligomer in cis-association at cell surface. A significant amount of CD9P-1 oligomer was observed on various cell types. We have shown that CD9P-1 self-associates independently from its association with tetraspanins. However, the expression level of CD9 or CD81 that associate directly and specifically with CD9P-1, positively modulates the cross-linking efficiency of CD9P-1. Thus, tetraspanins can play a role on CD9P-1 oligomerization status.     

Multiple levels of interactions within the tetraspanin web

The tetraspanin web refers to a network of molecular interactions involving tetraspanins and other molecules. Inside the tetraspanin web, small primary complexes containing only one tetraspanin and one specific partner molecule such as CD151/alpha3beta1 integrin and CD9/CD9P-1 (FPRP) can be observed under particular conditions. Here we demonstrate that when cells are lysed with Brij97, the tetraspanins CD151 and CD9 allow and/or stabilize the interaction of their partner molecules with other tetraspanins and that their two partners associate under conditions maintaining tetraspanin/tetraspanin interactions. The tetraspanins were also found to partition into a detergent-resistant membrane environment to which the integrin alpha3beta1 was relocalized upon expression of CD151.     

A novel cysteine cross-linking method reveals a direct association between claudin-1 and tetraspanin CD9

Tetraspanins serve as molecular organizers of multiprotein microdomains in cell membranes. Hence to understand functions of tetraspanin proteins, it is critical to identify laterally interacting partner proteins. Here we used a novel technical approach involving exposure and cross-linking of membrane-proximal cysteines coupled with LC-MS/MS protein identification. In this manner we identified nine potential tetraspanin CD9 partners, including claudin-1. Chemical cross-linking yielded a CD9-claudin-1 heterodimer, thus confirming direct association and adding claudin-1 to the short list of proteins that can directly associate with CD9. Interaction of CD9 (and other tetraspanins) with claudin-1 was supported by subcellular colocalization and was confirmed in multiple cell lines, although other claudins (claudin-2, -3, -4, -5, and -7) associated to a much lesser extent. Moreover claudin-1 was distributed very similarly to CD9 in sucrose gradients and, like CD9, was released from A431 and A549 cells upon cholesterol depletion. These biochemical features of claudin-1 are characteristic of tetraspanin microdomain proteins. Although claudins are major structural components of intercellular tight junctions, CD9-claudin-1 complexes did not reside in tight junctions, and depletion of key tetraspanins (CD9 and CD151) by small interfering RNA had no effect on paracellular permeability. However, tetraspanin depletion did cause a marked decrease in the stability of newly synthesized claudin-1. In conclusion, these results (a) validate a technical approach that appears to be particularly well suited for identifying protein partners directly associated with tetraspanins or with other proteins that contain membrane-proximal cysteines and (b) provide insight into how non-junctional claudins may be regulated in the context of tetraspanin-enriched microdomains.     

The major CD9 and CD81 molecular partner. Identification and characterization of the complexes

By associating with specific partner molecules and with each other, the tetraspanins are thought to assemble multimolecular complexes that may be especially relevant with respect to metastasis. We have previously identified a 135-kDa molecule (CD9P-1) as a major molecular partner of CD9 in cancer cell lines. This molecule was identified, after immunoaffinity purification and mass spectrometry analysis, as the protein encoded by the KIAA1436 gene and the human ortholog of a rat protein known as FPRP. Cross-linking experiments detected a complex of the size of CD9 plus CD9P-1, showing that these glycoproteins directly associate with each other, probably in the absence of any other molecule. The use of chimeric CD9/CD82 molecules revealed the role of the second half of CD9, comprising the large extracellular loop and the fourth transmembrane domain. CD9P-1 was also shown to form separate complexes with CD81 and with an unidentified 175-kDa molecule. It also associated with other tetraspanins under conditions maintaining tetraspanin/tetraspanin interactions. The identification of a protein strongly linked to the tetraspanin web and the production of a specific monoclonal antibody will help to further characterize the role of this "web" under physiological and pathological conditions.     

Dynamic regulation of a GPCR-tetraspanin-G protein complex on intact cells: central role of CD81 in facilitating GPR56-Galpha q/11 association

By means of a variety of intracellular scaffolding proteins, a vast number of heterotrimeric G protein-coupled receptors (GPCRs) may achieve specificity in signaling through a much smaller number of heterotrimeric G proteins. Members of the tetraspanin family organize extensive complexes of cell surface proteins and thus have the potential to act as GPCR scaffolds; however, tetraspanin-GPCR complexes had not previously been described. We now show that a GPCR, GPR56/TM7XN1, and heterotrimeric G protein subunits, Galpha(q), Galpha(11), and Gbeta, associate specifically with tetraspanins and CD81, but not with other tetraspanins. CD9 Complexes of GPR56 with CD9 and CD81 remained intact when fully solubilized and were resistant to cholesterol depletion. Hence they do not depend on detergent-insoluble, raft-like membrane microdomains for stability. A central role for CD81 in promoting or stabilizing a GPR56-CD81-Galpha(q/11) complex was revealed by CD81 immunodepletion and reexpression experiments. Finally, antibody engagement of cell surface CD81 or cell activation with phorbol ester revealed two distinct mechanisms by which GPR56-CD81-Galpha(q/11) complexes can be dynamically regulated. These data reveal a potential role for tetraspanins CD9 and CD81 as GPCR scaffolding proteins.     

The Tetraspanins CD9 and CD81 Regulate CD9P1-Induced Effects on Cell Migration

CD9P-1 is a cell surface protein with immunoglobulin domains and an unknown function that specifically associates with tetraspanins CD9 and CD81. Overexpression of CD9P-1 in HEK-293 cells induces dramatic changes in cell spreading and migration on various matrices. Experiments using time-lapse videomicroscopy revealed that CD9P-1 expression has led to higher cell motility on collagen I but lower motility on fibronectin through a β1-integrins dependent mechanism. On collagen I, the increase in cell motility induced by CD9P-1 expression was found to involve integrin α2β1 and CD9P-1 was observed to associate with this collagen receptor. The generation of CD9P-1 mutants demonstrated that the transmembrane and the cytoplasmic domains are necessary for inducing effects on cell motility. On the other hand, expression of tetraspanins CD9 or CD81 was shown to reverse the effects of CD9P-1 on cell motility on collagen I or fibronectin with a concomitant association with CD9P-1. Thus, the ratio of expression levels between CD9P-1 and its tetraspanin partners can regulate cell motility.     

Glycosylation status of the membrane protein CD9P-1

The membrane protein CD9P-1 is a major component of the tetraspanin web, a network of molecular interactions in the plasma membrane, in which it specifically associates with tetraspanins CD9 and CD81. The various functional effects of CD9 and CD81 may be related to their partners. Thus, we have addressed the characterization of the CD9P-1 glycosylation using stably transfected HEK-293 cells. After immunoprecipitation, CD9P-1 was subjected to enzymatic PNGase F cleavage of N-glycans, resulting in Asn to Asp conversion and increase in 1 mass unit. Thus, following protease digestion, deglycosylated peptides were selectively identified by high mass accuracy FTICR-MS, using this conversion as a signature. This has demonstrated that all nine potential N-glycosylation sites were actually engaged. On the other hand, the N-glycan structures were determined combining chemical derivatization and exoglycosidase digestions followed by MALDI-TOF MS, ESI-MS/MS, and GC-MS analysis. CD9P-1 was shown to exhibit more than 40 different N-glycans, essentially composed of complex and high mannose-type structures. Finally, 2-D PAGE and lectino-blot analyses have revealed the presence of at least 17 glycosylated isoforms of CD9P-1 at cell surface. All CD9P-1 isoforms associate with CD9 leading to additional level of complexity of this primary complex in the tetraspanin web.     

The Ig Domain Protein CD9P-1 Down-regulates CD81 Ability to Support Plasmodium yoelii Infection

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria natural infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that CD81 is required on hepatocytes for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 belongs to the tetraspanin superfamily of transmembrane proteins. By interacting with each other and with other transmembrane proteins, tetraspanins may play a role in the lateral organization of membrane proteins. In this study, we investigated the role of the two major molecular partners of CD81 in hepatocytic cells, CD9P-1/EWI-F and EWI-2, two transmembrane proteins belonging to a novel subfamily of immunoglobulin proteins. We show that CD9P-1 silencing increases the host cell susceptibility to P. yoelii sporozoite infection, whereas EWI-2 knock-down has no effect. Conversely, overexpression of CD9P-1 but not EWI-2 partially inhibits infection. Using CD81 and CD9P-1 chimeric molecules, we demonstrate the role of transmembrane regions in CD81-CD9P-1 interactions. Importantly, a CD9P-1 chimera that no longer associates with CD81 does not affect infection. Based on these data, we conclude that CD9P-1 acts as a negative regulator of P. yoelii infection by interacting with CD81 and regulating its function.     

Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathione S-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events.     

A new class of tetraspanins in fungi

Tetraspanins are animal proteins involved in membrane complexes that are involved in cell adhesion, differentiation, and motility. The PLS1 gene from rice blast fungus Magnaporthe grisea encodes a protein (Pls1p) structurally related to tetraspanins that is required for pathogenicity. In Botrytis cinerea public sequences, we identified an EST homologous to PLS1. Using degenerated oligonucleotides, we amplified sequences homologous to PLS1 in fungi Colletotrichum lindemuthianum and Neurospora crassa. Analysis of N. crassa and M. grisea genome sequences revealed the presence of a single tetraspanin gene. Thus, fungi differ from animals, which contain between 20 and 37 paralogous tetraspanin genes. Fungal proteins encoded by BcPLS1, ClPLS1, and NcPLS1 display all the structural hallmarks of tetraspanins (predicted topology with four transmembrane domains, extra- and intracellular loops; conserved cysteine-based patterns in second extracellular loop). Phylogenetic analysis suggests that these genes define a new family of orthologous genes encoding fungal-specific tetraspanins.     

Characterization of integrin-tetraspanin adhesion complexes: role of tetraspanins in integrin signaling.

Tetraspanins (or proteins from the transmembrane 4 superfamily, TM4SF) form membrane complexes with integrin receptors and are implicated in integrin-mediated cell migration. Here we characterized cellular localization, structural composition, and signaling properties of alpha3beta1-TM4SF adhesion complexes. Double-immunofluorescence staining showed that various TM4SF proteins, including CD9, CD63, CD81, CD82, and CD151 are colocalized within dot-like structures that are particularly abundant at the cell periphery. Differential extraction in conjunction with chemical cross-linking indicated that the cell surface fraction of alpha3beta1-TM4SF protein complexes may not be directly linked to the cytoskeleton. However, in cells treated with cytochalasin B alpha3beta1-TM4SF protein complexes are relocated into intracellular vesicles suggesting that actin cytoskeleton plays an important role in the distribution of tetraspanins into adhesion structures. Talin and MARCKS are partially codistributed with TM4SF proteins, whereas vinculin is not detected within the tetraspanin-containing adhesion structures. Attachment of serum-starved cells to the immobilized anti-TM4SF mAbs induced dephosphorylation of focal adhesion kinase (FAK). On the other hand, clustering of tetraspanins in cells attached to collagen enhanced tyrosine phosphorylation of FAK. Furthermore, ectopic expression of CD9 in fibrosarcoma cells affected adhesion-induced tyrosine phosphorylation of FAK, that correlated with the reorganization of the cortical actin cytoskeleton. These results show that tetraspanins can modulate integrin signaling, and point to a mechanism by which TM4SF proteins regulate cell motility.     

Palmitoylation supports assembly and function of integrin-tetraspanin complexes

As observed previously, tetraspanin palmitoylation promotes tetraspanin microdomain assembly. Here, we show that palmitoylated integrins (alpha3, alpha6, and beta4 subunits) and tetraspanins (CD9, CD81, and CD63) coexist in substantially overlapping complexes. Removal of beta4 palmitoylation sites markedly impaired cell spreading and signaling through p130Cas on laminin substrate. Also in palmitoylation-deficient beta4, secondary associations with tetraspanins (CD9, CD81, and CD63) were diminished and cell surface CD9 clustering was decreased, whereas core alpha6beta4-CD151 complex formation was unaltered. There is also a functional connection between CD9 and beta4 integrins, as evidenced by anti-CD9 antibody effects on beta4-dependent cell spreading. Notably, beta4 palmitoylation neither increased localization into "light membrane" fractions of sucrose gradients nor decreased solubility in nonionic detergents-hence it does not promote lipid raft association. Instead, palmitoylation of beta4 (and of the closely associated tetraspanin CD151) promotes CD151-alpha6beta4 incorporation into a network of secondary tetraspanin interactions (with CD9, CD81, CD63, etc.), which provides a novel framework for functional regulation.     

The tetraspanin web modulates immune-signalling complexes

The tetraspanin web represents a new concept of molecular interactions in the immune system. Whereas most surface immune-modulating molecules involve receptor-ligand interactions, tetraspanins associate with partner proteins and facilitate their lateral positioning in the membrane. Moreover, the same tetraspanin molecule can associate with different proteins depending on the cell type. Most importantly, members of this family tend to associate with each other, together with their partners, in membrane microdomains that provide a scaffold for the transmission of external stimuli to intracellular-signalling components.     

DHHC2 affects palmitoylation, stability, and functions of tetraspanins CD9 and CD151

Although palmitoylation markedly affects tetraspanin protein biochemistry and functions, relevant palmitoylating enzymes were not known. There are 23 mammalian "DHHC" (Asp-His-His-Cys) proteins, which presumably palmitoylate different sets of protein substrates. Among DHHC proteins tested, DHHC2 best stimulated palmitoylation of tetraspanins CD9 and CD151, whereas inactive DHHC2 (containing DH-->AA or C-->S mutations within the DHHC motif) failed to promote palmitoylation. Furthermore, DHHC2 associated with CD9 and CD151, but not other cell surface proteins, and DHHC2 knockdown diminished CD9 and CD151 palmitoylation. Knockdown of six other Golgi-resident DHHC proteins (DHHC3, -4, -8, -17, -18, and -21) had no effect on CD9 or CD151. DHHC2 selectively affected tetraspanin palmitoylation, but not the palmitoylations of integrin beta4 subunit and bulk proteins visible in [(3)H]palmitate-labeled whole cell lysates. DHHC2-dependent palmitoylation also had multiple functional effects. First, it promoted physical associations between CD9 and CD151, and between alpha3 integrin and other proteins. Second, it protected CD151 and CD9 from lysosomal degradation. Third, the presence of DHHC2, but not other DHHC proteins, shifted cells away from a dispersed state and toward increased cell-cell contacts.     

Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web

Tetraspanins regulate cell migration, sperm–egg fusion, and viral infection. Through interactions with one another and other cell surface proteins, tetraspanins form a network of molecular interactions called the tetraspanin web. In this study, we use single-molecule fluorescence microscopy to dissect dynamics and partitioning of the tetraspanin CD9. We show that lateral mobility of CD9 in the plasma membrane is regulated by at least two modes of interaction that each exhibit specific dynamics. The majority of CD9 molecules display Brownian behavior but can be transiently confined to an interaction platform that is in permanent exchange with the rest of the membrane. These platforms, which are enriched in CD9 and its binding partners, are constant in shape and localization. Two CD9 molecules undergoing Brownian trajectories can also codiffuse, revealing extra platform interactions. CD9 mobility and partitioning are both dependent on its palmitoylation and plasma membrane cholesterol. Our data show the high dynamic of interactions in the tetraspanin web and further indicate that the tetraspanin web is distinct from raft microdomains.     

The single subunit transmembrane E3 ligase gene related to anergy in lymphocytes (GRAIL) captures and then ubiquitinates transmembrane proteins across the cell membrane

The ubiquitin E3 ligase gene related to anergy in lymphocytes (GRAIL) (Rnf128) is a type 1 transmembrane protein that induces T cell anergy through the ubiquitination activity of its cytosolic RING finger. GRAIL also contains an equally large luminal region consisting primarily of an uncharacterized protease-associated (PA) domain. Using two-hybrid technology to screen for proteins that bound the PA domain we identified CD151, a member of the tetraspanin family of membrane proteins. GRAIL bound to the luminal/extracellular portion of both CD151 and the related tetraspanin CD81 using its PA domain, which promoted ubiquitination of cytosolic lysine residues. GRAIL exhibited specificity for lysines only within the tetraspanin amino terminus even in the presence of other cytosolic lysine residues in the substrate. GRAIL-mediated ubiquitination promoted proteasomal degradation and cell surface down-regulation of tetraspanins via Lys-48 linkages. As a result, the juxtaposition of PA and RING finger domains across a lipid bilayer facilitates the capture of transmembrane substrates for subsequent ubiquitination. These findings identify for the first time a single subunit E3 ligase containing a substrate-binding domain spatially restricted by a membrane from its E2 recruitment domain as well as an E3 ligase for members of the tetraspanin family.     

Tetraspanins in the humoral immune response

The tetraspanins represent a large superfamily of four-transmembrane proteins that are expressed on all nucleated cells. Tetraspanins play a prominent role in the organization of the plasma membrane by co-ordinating the spatial localization of transmembrane proteins and signalling molecules into 'tetraspanin microdomains'. In immune cells, tetraspanins interact with key leucocyte receptors [including MHC molecules, integrins, CD4/CD8 and the BCR (B-cell receptor) complex] and as such can modulate leucocyte receptor activation and downstream signalling pathways. There is now ample evidence that tetraspanins on B-lymphocytes are important in controlling antibody production. The tetraspanin CD81 interacts with the BCR complex and is critical for CD19 expression and IgG production, whereas the tetraspanin CD37 inhibits IgA production and is important for IgG production. By contrast, the tetraspanins CD9, Tssc6 and CD151 appear dispensable for humoral immune responses. Thus individual tetraspanin family members have specific functions in B-cell biology, which is evidenced by recent studies in tetraspanin-deficient mice and humans. The present review focuses on tetraspanins expressed by B-lymphocytes and discusses novel insights into the function of tetraspanins in the humoral immune response.     

EWI-2 regulates alpha3beta1 integrin-dependent cell functions on laminin-5

EWI-2, a cell surface immunoglobulin SF protein of unknown function, associates with tetraspanins CD9 and CD81 with high stoichiometry. Overexpression of EWI-2 in A431 epidermoid carcinoma cells did not alter cell adhesion or spreading on laminin-5, and had no effect on reaggregation of cells plated on collagen I (alpha2beta1 integrin ligand). However, on laminin-5 (alpha3beta1 integrin ligand), A431 cell reaggregation and motility functions were markedly impaired. Immunodepletion and reexpression experiments revealed that tetraspanins CD9 and CD81 physically link EWI-2 to alpha3beta1 integrin, but not to other integrins. CD81 also controlled EWI-2 maturation and cell surface localization. EWI-2 overexpression not only suppressed cell migration, but also redirected CD81 to cell filopodia and enhanced alpha3beta1-CD81 complex formation. In contrast, an EWI-2 chimeric mutant failed to suppress cell migration, redirect CD81 to filopodia, or enhance alpha3beta1-CD81 complex formation. These results show how laterally associated EWI-2 might regulate alpha3beta1 function in disease and development, and demonstrate how tetraspanin proteins can assemble multiple nontetraspanin proteins into functional complexes.