Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins.
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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. (+info)
Development of human lymphohematopoietic stem and progenitor cells defined by expression of CD34 and CD81.
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In this study, cord blood CD34(+) cells expressed CD81, a member of the transmembrane 4 superfamily, and were classified into 3 subpopulations on the basis of their expression levels: CD34(+)CD81(+), CD34(low)CD81(+), and CD34(+)CD81(high). The lymphohematopoietic activity of each subpopulation was then examined by using suspension and clonogenic cultures for hematopoietic potential, coculture with MS-5 cells for B-cell potential, organ cultures of thymus lobes from nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) fetal mice, coculture with stromal cells derived from NOD/SCID fetal-mouse liver tissue for natural killer (NK) cell and mast cell potentials, and xenotransplantation into NOD/SCID mice for long-term repopulating (LTR) ability. CD34(+)CD81(+) cells represented a heterogeneous population that had all the lymphohematopoietic activities, including NOD/SCID mouse-repopulating ability. CD34(low)CD81(+) cells were enriched in erythroid, megakaryocytic, and NK lineage potentials but had lost T-cell and B-cell potentials and LTR ability. The CD34(+)CD81(high) fraction was depleted of most lymphohematopoietic potentials except NK cell and mast cell potentials. Thus, along the differentiation cascade from CD34(+)CD81(+) lymphohematopoietic stem cells, an up-regulation of CD81 or a down-regulation of CD34 results in a change in lymphohematopoietic properties. CD81 may serve as a marker for defining developmental stages of lymphohematopoietic stem cells. (Blood. 2001;97:3755-3762) (+info)
Influence of beta1 integrins on epidermal squamous cell carcinoma formation in a transgenic mouse model: alpha3beta1, but not alpha2beta1, suppresses malignant conversion.
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Although aberrant integrin expression has been documented in many epithelial tumors, little is known about how integrins influence neoplastic progression. To examine this issue, transgenic mice in which the alpha2beta1 or alpha3beta1 integrin was expressed in the suprabasal epidermal layers via the involucrin promoter were subjected to skin carcinogenesis. Equal numbers of benign squamous papillomas were observed in transgenic and wild-type animals. However, the frequency of conversion of papillomas to malignant squamous cell carcinomas was much lower in alpha3beta1 transgenic than in alpha2beta1 transgenic and wild-type mice. No differences were observed in apoptosis or in the expression of endogenous integrins in transgenic and wild-type papillomas. However, alpha3beta1 transgenic papillomas displayed a diminished proliferative capacity and were more highly differentiated as judged by BrdUrd incorporation and keratin 10 expression, respectively, than alpha2beta1 transgenic and wild-type papillomas. Two proteins that associate with alpha3beta1 and not alpha2beta1 are extracellular matrix metalloproteinase inducer and CD81. Extracellular matrix metalloproteinase inducer expression correlated inversely with the degree of differentiation in normal epidermis and in transgenic and wild-type papillomas. Up-regulation of CD81 was observed in 100% of wild-type and 88% of alpha2beta1 transgenic papillomas but in only 25% of alpha3beta1 transgenic papillomas. CD81 was undetectable in untreated epidermis and strongly expressed in all transgenic and wild-type squamous cell carcinomas. Our results demonstrate that the alpha3beta1 integrin can suppress malignant conversion, and that the mechanism may involve CD81. (+info)
Functional analysis of hepatitis C virus E2 glycoproteins and virus-like particles reveals structural dissimilarities between different forms of E2.
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Structure-function analysis of the hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, has been difficult due to the unavailability of HCV virions. Truncated soluble forms of E2 have been used as models to study virus interaction with the putative HCV receptor CD81, but they may not fully mimic E2 structures on the virion. Here, we compared the CD81-binding characteristics of truncated E2 (E2(660)) and full-length (FL) E1E2 complex expressed in mammalian cells, and of HCV virus-like particles (VLPs) generated in insect cells. All three glycoprotein forms interacted with human CD81 in an in vitro binding assay, allowing us to test a panel of well-characterized anti-E2 monoclonal antibodies (MAbs) for their ability to inhibit the glycoprotein-CD81 interaction. MAbs specific for E2 amino acid (aa) regions 396-407, 412-423 and 528-535 blocked binding to CD81 of all antigens tested. However, MAbs specific for regions 432-443, 436-443 and 436-447 inhibited the interaction of VLPs, but not of E2(660) or the FL E1E2 complex with CD81, indicating the existence of structural differences amongst the E2 forms. These findings underscore the need to carefully select an appropriate ligand for structure-function analysis. (+info)
Structure of the tetraspanin main extracellular domain. A partially conserved fold with a structurally variable domain insertion.
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The tetraspanin family of membrane glycoproteins is involved in the regulation of cellular development, proliferation, activation, and mobility. We have attempted to predict the structural features of the large extracellular domain of tetraspanins (EC2), which is very important in determining their functional specificity. The tetraspanin EC2 is composed of two subdomains: a conserved three-helix subdomain and a variable secondary structure subdomain inserted within the conserved subdomain. The occurrence of key disulphide bridges and other invariant residues leads to a conserved relative topology of both subdomains and also suggests a structural classification of tetraspanins. Using the CD81 EC2 structure as a template, the structures of two other EC2s were predicted by homology modeling and indicate a conserved shape, in which the variable subdomain is located at one side of the structure. The conserved and variable subdomains might contain sites that correspond, respectively, to common and specific interactions of tetraspanins. The tetraspanin EC2 seems to correspond to a new scheme of fold conservation/variability among proteins, namely the insertion of a structurally variable subdomain within an otherwise conserved fold. (+info)
EWI-2 is a major CD9 and CD81 partner and member of a novel Ig protein subfamily.
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A novel Ig superfamily protein, EWI-2, was co-purified with tetraspanin protein CD81 under relatively stringent Brij 96 detergent conditions and identified by mass spectrometric protein sequencing. EWI-2 associated specifically with CD9 and CD81 but not with other tetraspanins or with integrins. Immunodepletion experiments indicated that EWI-2-CD9/CD81 interactions are highly stoichiometric, with approximately 70% of CD9 and CD81 associated with EWI-2 in an embryonic kidney cell line. The EWI-2 molecule was covalently cross-linked (in separate complexes) to both CD81 and CD9, suggesting that association is direct. EWI-2 is part of a novel Ig subfamily that includes EWI-F (F2alpha receptor regulatory protein (FPRP), CD9P-1), EWI-3 (IgSF3), and EWI-101 (CD101). All four members of this Ig subfamily contain a Glu-Trp-Ile (EWI) motif not seen in other Ig proteins. As shown previously, the EWI-F molecule likewise forms highly proximal, specific, and stoichiometric complexes with CD9 and CD81. Human and murine EWI-2 protein sequences are 91% identical, and transcripts in the two species are expressed in virtually every tissue tested. Thus, EWI-2 potentially contributes to a variety of CD9 and CD81 functions seen in different cell and tissue types. (+info)
The dynamics of hepatitis C virus binding to platelets and 2 mononuclear cell lines.
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Hepatitis C virus (HCV) binds to platelets in chronically infected patients where free HCV constitutes only about 5% of total circulating virus. Free HCV preferentially binds to human mononuclear cell lines but free and complexed virus binds equally to platelets. The extent of free HCV binding to human Molt-4 T cells (which express CD81) and to human promonocytic U937 cells or to platelets (which do not express CD81) was similar. The binding of free HCV to the cell lines was saturated at a virus dose of 1 IU HCV RNA per cell but binding to platelets was not saturable. Human anti-HCV IgG, but not anti-CD81, markedly inhibited HCV binding to target cells in a dose-dependent manner. Human antibodies to HCV hypervariable region 1 of E2 glycoprotein partially inhibited viral binding to target cells. Recombinant E2 also inhibited viral binding to target cells in a dose-dependent manner, with the efficacy of this decreasing in the rank order of Molt-4 cells more than U937 cells more than platelets. In contrast to HCV, recombinant E2 bound to Molt-4 cells to an extent markedly greater than that apparent with U937 cells or platelets. These results suggest that the binding of HCV to blood cells is mediated by multiple cell surface receptors and that recombinant E2 binding may not be representative of the interaction of the intact virus with target cells. (+info)
PGRL is a major CD81-associated protein on lymphocytes and distinguishes a new family of cell surface proteins.
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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. (+info)