Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins

Translocation of conventional protein kinases C (PKCs) to the plasma membrane leads to their specific association with transmembrane-4 superfamily (TM4SF; tetraspanin) proteins (CD9, CD53, CD81, CD82, and CD151), as demonstrated by reciprocal co-immunoprecipitation and covalent cross-linking experiments. Although formation and maintenance of TM4SF-PKC complexes are not dependent on integrins, TM4SF proteins can act as linker molecules, recruiting PKC into proximity with specific integrins. Previous studies showed that the extracellular large loop of TM4SF proteins determines integrin associations. In contrast, specificity for PKC association probably resides within cytoplasmic tails or the first two transmembrane domains of TM4SF proteins, as seen from studies with chimeric CD9 molecules. Consistent with a TM4SF linker function, only those integrins (alpha(3)beta(1), alpha(6)beta(1), and a chimeric "X3TC5" alpha(3) mutant) that associated strongly with tetraspanins were found in association with PKC. We propose that PKC-TM4SF-integrin structures represent a novel type of signaling complex. The simultaneous binding of TM4SF proteins to the extracellular domains of the integrin alpha(3) subunit and to intracellular PKC helps to explain why the integrin alpha3 extracellular domain is needed for both intracellular PKC recruitment and PKC-dependent phosphorylation of the alpha(3) integrin cytoplasmic tail.     

Evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts

Recent literature suggests that tetraspanin proteins (transmembrane 4 superfamily; TM4SF proteins) may associate with each other and with many other transmembrane proteins to form large complexes that sometimes may be found in lipid rafts. Here we show that prototype complexes of CD9 or CD81 (TM4SF proteins) with alpha(3)beta(1) (an integrin) and complexes of CD63 (a TM4SF protein) with phosphatidylinositol 4-kinase (PtdIns 4-K) may indeed localize within lipid raft-like microdomains, as seen by three different criteria. First, these complexes localize to low density light membrane fractions in sucrose gradients. Second, CD9 and alpha(3) integrin colocalized with ganglioside GM1 as seen by double staining of fixed cells. Third, CD9-alpha3beta1 and CD81-alpha3beta1 complexes were shifted to a higher density upon cholesterol depletion from intact cells or cell lysate. However, CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K complex formation itself was not dependent on localization into raftlike lipid microdomains. These complexes did not require cholesterol for stabilization, were maintained within well solubilized dense fractions from sucrose gradients, were stable at 37 degrees C, and were small enough to be included within CL6B gel filtration columns. In summary, prototype TM4SF protein complexes (CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K) can be solubilized as discrete units, independent of lipid microdomains, although they do associate with microdomains resembling lipid rafts.     

Role of transmembrane 4 superfamily (TM4SF) proteins CD9 and CD81 in muscle cell fusion and myotube maintenance.

The role of transmembrane 4 superfamily (TM4SF) proteins during muscle cell fusion has not been investigated previously. Here we show that the appearance of TM4SF protein, CD9, and the formation of CD9-beta1 integrin complexes were both regulated in coordination with murine C2C12 myoblast cell differentiation. Also, anti-CD9 and anti-CD81 monoclonal antibodies substantially inhibited and delayed conversion of C2C12 cells to elongated myotubes, without affecting muscle-specific protein expression. Studies of the human myoblast-derived RD sarcoma cell line further demonstrated that TM4SF proteins have a role during muscle cell fusion. Ectopic expression of CD9 caused a four- to eightfold increase in RD cell syncytia formation, whereas anti-CD9 and anti-CD81 antibodies markedly delayed RD syncytia formation. Finally, anti-CD9 and anti-CD81 monoclonal antibodies triggered apoptotic degeneration of C2C12 cell myotubes after they were formed. In summary, TM4SF proteins such as CD9 and CD81 appear to promote muscle cell fusion and support myotube maintenance.     

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.     

Characterization of novel complexes on the cell surface between integrins and proteins with 4 transmembrane domains (TM4 proteins).

Here we identified several new integrin/TM4 protein complexes on the cell surface. By immunoprecipitation using nonstringent conditions, and by reciprocal immunoprecipitation, we found that alpha 3 beta 1 and alpha 6 beta 1 integrins but not alpha 2 beta 1, alpha 5 beta 1, or alpha 6 beta 4 integrins associated with CD9 and CD81 in alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, alpha 6 beta 1/CD81, and alpha 6 beta 1/CD9 complexes. Also, cross-linking experiments established that alpha 3 beta 1/CD81, alpha 3 beta 1/CD9, and alpha 3 beta 1/CD63 associations occur on the surface of intact cells and suggested that a critical interaction site is located within extracellular domains. Cross-linking in conjunction with reimmunoprecipitation indicated that larger multi-component alpha 3 beta 1/TM4/TM4 complexes (alpha 3 beta 1/CD9/CD63, alpha 3 beta 1/CD81/CD63, and alpha 3 beta 1/CD9/CD81) also could be detected on the cell surface. Immunofluorescent staining showed redistribution of alpha 3 beta 1/TM4 complexes toward the periphery of cells plated on various extracellular matrix substrates and also showed that these complexes were localized in cell footprints. Staining of human tissues yielded additional results consistent with co-localization of alpha 3 beta 1 and CD9, CD63, and CD81 proteins. In conclusion we suggest that the prevalence of integrin/TM4 complexes in diverse cellular environments is indicative of their general physiological importance.     

PGRL is a major CD81-associated protein on lymphocytes and distinguishes a new family of cell surface proteins.

CD81 exerts a range of interesting effects on T cells including early thymocyte differentiation, LFA-1 activation, and provision of costimulation. To better understand the mechanisms by which CD81 influences T cell function we evaluated CD81 molecular complexes on T cells. The most prominent CD81-associated cell surface protein on thymocytes as well as a number of T cell and B cell lines has an apparent molecular mass of 75 kDa. The 75-kDa protein was purified and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry followed by postsource-decay profiling. p75 is a novel type I transmembrane protein of the Ig superfamily which is most similar to FPRP. We cloned and sequenced both human and mouse PG regulatory-like protein (PGRL) and characterized mouse PGRL expression in both lymphocytes and nonlymphoid tissues. The discovery of PGRL allows for the clustering of a small family of related proteins including PGRL, FPRP, V7/CD101, and IGSF3. Expression constructs containing various domains of PGRL with an epitope tag were coexpressed with CD81 and used to determine that the interaction of CD81 with PGRL requires the membrane distal Ig3-Ig4 domains of PGRL. Although it remains to be determined whether PGRL possesses PG regulatory functions, transwell chamber experiments show that PGs and CD81 coordinately regulate T cell motility.     

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.     

Direct extracellular contact between integrin alpha(3)beta(1) and TM4SF protein CD151

Previously we established that the alpha(3)beta(1) integrin shows stable, specific, and stoichiometric association with the TM4SF (tetraspannin) protein CD151. Here we used a membrane impermeable cross-linking agent to show a direct association between extracellular domains of alpha(3)beta(1) and CD151. The alpha(3)beta(1)-CD151 association site was then mapped using chimeric alpha(6)/alpha(3) integrins and CD151/NAG2 TM4SF proteins. Complex formation required an extracellular alpha(3) site (amino acids (aa) 570-705) not previously known to be involved in specific integrin contacts with other proteins and a region (aa 186-217) within the large extracellular loop of CD151. Notably, the anti-CD151 monoclonal antibody TS151r binding epitope, previously implicated in alpha(3) integrin association, was mapped to the same region of CD151 (aa 186-217). Finally, we demonstrated that both NH(2)- and COOH-terminal domains of CD151 are located on the inside of the plasma membrane, thus confirming a long suspected model of TM4SF protein topology.     

Association of the Tetraspan Protein CD9 with Integrins on the Surface of S-16 Schwann Cells

Abstract: The cell surface glycoprotein CD9 is a member of a group of proteins known as the "tetraspan" or "transmembrane 4 superfamily." Previous work with non-neural cells has shown that CD9 associates in cis with integrins and small GTP-binding proteins on the cell surface. To extend our recent findings showing that perturbation of CD9 alters Schwann cell adhesion, proliferation, and migration, as well as neurite outgrowth in sympathetic neurons, we have searched for CD9-associated proteins in S-16 Schwann cells. We demonstrate here that CD9 is specifically coprecipitated from S-16 cell extracts by antibodies against integrins α3, α6, and β1. In addition, double immunofluorescence labeling and co-capping experiments indicate that CD9 is specifically colocalized with these integrins on the cell membrane of S-16 Schwann cells.     

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface.In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.     

Highly Stoichiometric, Stable, and Specific Association of Integrin α3β1 with CD151 Provides a Major Link to Phosphatidylinositol 4-Kinase, and May Regulate Cell Migration

Here we describe an association between α3β1 integrin and transmembrane-4 superfamily (TM4SF) protein CD151. This association is maintained in relatively stringent detergents and thus is remarkably stable in comparison with previously reported integrin–TM4SF protein associations. Also, the association is highly specific (i.e., observed in vitro in absence of any other cell surface proteins), and highly stoichiometric (nearly 90% of α3β1 associated with CD151). In addition, α3β1 and CD151 appeared in parallel on many cell lines and showed nearly identical skin staining patterns. Compared with other integrins, α3β1 exhibited a considerably higher level of associated phosphatidylinositol-4-kinase (PtdIns 4-kinase) activity, most of which was removed upon immunodepletion of CD151. Specificity for CD151 and PtdIns 4-kinase association resided in the extracellular domain of α3β1, thus establishing a novel paradigm for the specific recruitment of an intracellular signaling molecule. Finally, antibodies to either CD151 or α3β1 caused a ~88–92% reduction in neutrophil motility in response to f-Met-Leu-Phe on fibronectin, suggesting an functionally important role of these complexes in cell migration.     

慢病毒介导的重组TM9SF1蛋白通过诱导自噬和内质网应激抑制293T细胞生长

九次跨膜超家族蛋白成员1（transmembrane 9 superfamily protein member 1,TM9SF1）在进化过程中高度保守,在人体组织和多种细胞系广泛表达。目前,关于该蛋白质的功能研究十分有限和初步。本研究采用慢病毒介导的TM9SF1表达系统,研究了重组TM9SF1蛋白的生化特点及其对细胞生长的调控作用。慢病毒感染的293T全细胞裂解液的蛋白质免疫印迹结果揭示,TM9SF1蛋白具有表观分子质量约为70 kD的单体及寡聚体两种主要形式;在室温及加热37℃时蛋白质相对稳定,随变性温度升高（56 ℃以上）逐渐失去其稳定性。CCK8法显示,与慢病毒空载体感染的293T细胞比较,TM9SF1慢病毒表达载体感染的293T细胞在感染2 d后增殖明显减缓（P<0.001）。Western印迹结果证明,过表达TM9SF1引起LC3Ⅱ表达明显上调,LC3Ⅱ/LC3Ⅰ比例升高,说明TM9SF1可引起293T细胞发生自噬。荧光实时定量PCR结果显示,过表达TM9SF1的293T细胞内质网应激标志分子CHOP、GADD34和XBP1（S）表达水平是对照细胞的3～4倍,提示发生了内质网应激反应。以上结果提示,TM9SF1具有抑制293T细胞生长的功能,该功能可能与其引起的内质网应激和自噬有关。这一结论将进一步加深对TM9SF1在细胞生长调控中的功能的认识。     

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.     

Osteoblast-enriched membrane protein IFITM5 regulates the association of CD9 with an FKBP11-CD81-FPRP complex and stimulates expression of interferon-induced genes

Osteoblasts are rich in interferon-inducible transmembrane protein 5 (IFITM5), the expression of which peaks around the early mineralization stage. This membrane protein directly associates with FK506 binding protein 11 (FKBP11). To examine the molecular function of IFITM5, we analyzed the protein interaction network around IFITM5–FKBP11. We found that FKBP11 was associated with CD81, which interacts with prostaglandin F2 receptor negative regulator (FPRP) and CD9; cumulatively, these associations result in the formation of a FKBP11–CD81–[FPRP/CD9] complex. However, CD9 dissociated from the complex following expression of Ifitm5, which also led to osteoblast-specific increased expression of 5 interferon-induced genes: bone marrow stromal cell antigen 2 (Bst2), interferon inducible protein 1 (Irgm), interferon-induced protein with tetratricopeptide repeats 3 (Ifit3), b(2)-microglobulin (B2m), and MHC (A.CA/J(H-2K-f) class I antigen gene. Induction of these genes likely resulted from dissociation of CD9 from the FKBP11–CD81–[FPRP/CD9] complex. Cumulatively, these results suggest that IFITM5 is involved not only in bone formation, but also in immune system activity.     

Importance of the major extracellular domain of CD9 and the epidermal growth factor (EGF)-like domain of heparin-binding EGF-like growth factor for up-regulation of binding and activity

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors. The membrane-anchored form of HB-EGF (proHB-EGF) is mitogenically active to neighboring cells as well as being a precursor of the soluble form. In addition to its mitogenic activity, proHB-EGF has the property of binding to diphtheria toxin (DT), serving as the specific receptor for DT. Tetramembrane-spanning protein CD9, a member of the TM4 superfamily, is physically associated with proHB-EGF at the cell surface and up-regulates both mitogenic and DT binding activities of proHB-EGF. To understand this up-regulation mechanism, we studied essential regions of both CD9 and proHB-EGF for up-regulation. Immunoprecipitation experiments revealed that not only CD9 but also other TM4 proteins including CD63, CD81, and CD82 associate with proHB-EGF on the cell surface. However, these TM4 proteins did not up-regulate DT binding activity of proHB-EGF. Transfection of a series of chimeric constructs comprising CD9 and CD81 showed that the major extracellular domain of CD9 is essential for up-regulation. Assays of DT binding activity and juxtacrine mitogenic activity of the deletion mutants of proHB-EGF and chimeric molecules, derived from proHB-EGF and TGF-alpha, showed that the essential domain of proHB-EGF for up-regulation is the EGF-like domain. These results indicate that the interaction of the extracellular domains of both molecules is important for up-regulation.     

Correlations between Transmembrane 4 L6 Family Member 5 (TM4SF5), CD151, and CD63 in Liver Fibrotic Phenotypes and Hepatic Migration and Invasive Capacities

Transmembrane 4 L6 family member 5 (TM4SF5) is overexpressed during CCl₄-mediated murine liver fibrosis and in human hepatocellular carcinomas. The tetraspanins form tetraspanin-enriched microdomains (TEMs) consisting of large membrane protein complexes on the cell surface. Thus, TM4SF5 may be involved in the signal coordination that controls liver malignancy. We investigated the relationship between TM4SF5-positive TEMs with liver fibrosis and tumorigenesis, using normal Chang hepatocytes that lack TM4SF5 expression and chronically TGFβ1-treated Chang cells that express TM4SF5. TM4SF5 expression is positively correlated with tumorigenic CD151 expression, but is negatively correlated with tumor-suppressive CD63 expression in mouse fibrotic and human hepatic carcinoma tissues, indicating cooperative roles of the tetraspanins in liver malignancies. Although CD151 did not control the expression of TM4SF5, TM4SF5 appeared to control the expression levels of CD151 and CD63. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface into late lysosomal membranes, presumably leading to terminating the tumor-suppressive functions of CD63. TM4SF5 could overcome the tumorigenic effects of CD151, especially cell migration and extracellular matrix (ECM)-degradation. Taken together, TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins on the cell surface, and could provide a promising therapeutic target for the treatment of liver malignancies.     

Normal development but differentially altered proliferative responses of lymphocytes in mice lacking CD81.

CD81 (TAPA-1) is a member of the transmembrane 4 superfamily (TM4SF) which is expressed on the cell surface of most cells of the body throughout their cellular differentiation. It has been recognized in several cell surface complexes of lymphocytes, suggesting that it may have diverse roles in lymphocyte development and activation regulation. Mice with a CD81 null mutation revealed normal T- and conventional B-cell development, although CD19 expression on B cells was dull and B-1 cells were reduced in number. However, both T and B cells of mutant mice exhibited strikingly enhanced proliferation in response to various types of stimuli. Interestingly, while proliferative responses of T cells following T-cell antigen receptor (TCR) engagement was enhanced in the absence of CD81, B-cell proliferation in response to B-cell antigen-receptor (BCR) cross-linking was severely impaired. Despite these altered proliferative responses, both tyrosine phosphorylation and intracellular calcium flux in response to cross-linking of cell surface antigen receptors were normal in mutant mice, reflecting apparently normal initial signaling of antigen receptors. In conclusion, though CD81 is not essential for normal T- and conventional B-cell development, it plays key roles in controlling lymphocyte homeostasis by regulating lymphocyte proliferation in distinct manners, dependent on the context of stimulation.     

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.     

Analysis of the CD151-alpha3beta1 integrin and CD151-tetraspanin interactions by mutagenesis

Transmembrane proteins of the tetraspanin superfamily are associated with various integrins and modulate their function. We performed mutagenesis analysis to establish structural requirements for the interaction of CD151 with the alpha3beta1 integrin and with other tetraspanins. Using a panel of CD151/CD9 chimeras and CD151 deletion mutants we show that the minimal region, which confers stable (e.g. Triton X-100-resistant) association of the tetraspanin with alpha3beta1, maps within the large extracellular loop (LECL) of CD151 (the amino acid sequence between residues Leu(149) and Glu(213)). Furthermore, the substitution of 11 amino acids (residues 195-205) from this region for a corresponding sequence from CD9 LECL or point mutations of cysteines in the conserved CCG and PXXCC motifs abolish the interaction. The removal of the LECL CD151 does not affect the association of the protein with other tetraspanins (e.g. CD9, CD81, CD63, and wild-type CD151). On the other hand, the mutation of the CCG motif selectively prevents the homotypic CD151-CD151 interaction but does not influence the association of the mutagenized CD151 with other tetraspanins. These results demonstrate the differences in structural requirements for the heterotypic and homotypic tetraspanin-tetraspanin interactions. Various deletions involving the small extracellular loop and the first three transmembrane domains prevent surface expression of the CD151 mutants but do not affect the CD151-alpha3beta1 interaction. The CD151 deletion mutants are accumulated in the endoplasmic reticulum and redirected to the lysosomes. The assembly of the CD151-alpha3beta1 complex occurs early during the integrin biosynthesis and precedes the interaction of CD151 with other tetraspanins. Collectively, these data show that the incorporation of CD151 into the "tetraspanin web" can be controlled at various levels by different regions of the protein.