Scott C. Todd

The tetraspanin superfamily: molecular facilitators.

A legacy of molecular evolution is the formation of gene families encoding proteins that often serve related functions. One such family gaining recent attention is the tetraspanin superfamily, whose membership has grown to nearly 20 known genes since its discovery in 1990. All encode cell‐surface proteins that span the membrane four times, forming two extracellular loops. Some of these genes are found in organisms as primitive as schistosomes and nematodes. Alternately known as the transmembrane 4 (TM4) superfamily or the TM4SF, 4TM, or terraspan family, we propose here that the name tetraspanins be used for the purpose of standardization. What do the tetraspanins do? Awaiting definitive functional studies, we can only put together pieces of a puzzle that has been built by raising antibodies against these proteins and looking at their distribution, associations, and functions. A brief overview indicates that some tetraspanins are found in virtually all tissues (CD81, CD82, CD9, CD63), whereas others are highly restricted, such as CD37 (B cells) or CD53 (lymphoid and myeloid cells). Many of these proteins have a flair for promiscuous associations with other molecules, including lineage‐specific proteins, integrins, and other tetraspanins. In terms of function, they are involved in diverse processes such as cell activation and proliferation, adhesion and motility, differentiation, and cancer. We propose that these functions may all relate to their ability to act as “molecular facilitators,” grouping specific cell‐surface proteins and thus increasing the formation and stability of functional signaling complexes.—Maecker, H. T., Todd, S. C., Levy, S. The tetraspanin superfamily: molecular facilitators. FASEB J. 11, 428–442 (1997)

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.

Expression and Function of Formyl Peptide Receptors on Human Fibroblast Cells

The migration of polymorphonuclear leukocytes from the blood to sites of infection in tissues is a hallmark of the innate immune response. Formylated peptides produced as a byproduct of bacterial protein synthesis are powerful chemoattractants for leukocytes. Formyl peptides bind to two different G protein-coupled receptors (formyl peptide receptor (FPR) and the low affinity formyl peptide receptor-like-1 (FPRL1)) to initiate a signal transduction cascade leading to cell activation and migration. Our analysis of expressed sequences from many cDNA libraries draws attention to the fact that FPRs are widely expressed in nonlymphoid tissues. Here we demonstrate that FPRs are expressed by normal human lung and skin fibroblasts and the human fibrosarcoma cell line HT-1080. The expression on fibroblasts of receptors for bacteria-derived peptides raises questions about the possible function of these receptors in nonleukocyte cells. We studied the function of FPRs on fibroblasts and find that stimulation with fMLP triggers dose-dependent migration of these cells. Furthermore, fMLP induces signal transduction including intracellular calcium flux and a transient increase in F-actin. The fMLP-induced adhesion and motility of fibroblasts on fibronectin require functional protein kinase C and phosphatidylinositol 3-kinase. This first report of a functional formyl peptide receptor in cells of fibroblast origin opens new possibilities for the role of fibroblasts in innate immune responses.

Sequences and expression of six new members of the tetraspanin/TM4SF family

Tetraspanins (or TM4SF) are expressed in a wide variety of species and regulate cell adhesion, migration, proliferation and differentiation. We have identified and sequenced six new members of the tetraspanin family, called Tspan-1-6, from human cDNA. Amino acid sequence analysis of the Tspans highlights conserved residues which may be critical to tetraspanin structure and function. The Tspans are differentially expressed in human tissues.

Small molecule inhibition of hepatitis C virus E2 binding to CD81

The hepatitis C virus (HCV) is a causal agent of chronic liver infection, cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. CD81 is a receptor for HCV envelope glycoprotein E2. Although the binding of HCV-E2 with CD81 is well documented the role of this interaction in the viral life cycle remains unclear. Host specificity and mutagenesis studies suggest that the helix D region of CD81 mediates binding to HCV-E2. Structural analysis of CD81 has enabled the synthesis of small molecules designed to mimic the space and hydrophobic features of the solvent-exposed face on helix D. Utilizing a novel bis-imidazole scaffold a series of over 100 compounds has been synthesized. Seven related, imidazole-based compounds were identified that inhibit binding of HCV-E2 to CD81. The inhibitory compounds have no short-term effect on cellular expression of CD81 or other tetraspanins, do not disrupt CD81 associations with other cell surface proteins, and bind reversibly to HCV-E2. These results provide an important proof of concept that CD81-based mimics can disrupt binding of HCV-E2 to CD81.

Anti-CD81 activates LFA-1 on T cells and promotes T cell-B cell collaboration.

CD81 is expressed on human T cells at all stages of development. CD81 is physically associated with CD4 and CD8 and antibodies against CD81 generate signals which influence thymocyte adhesion and proliferation. Here we evaluate the function of CD81 on mature T cells. We employ a system in which B cells present superantigen to autologous T cells and find that anti‐CD81 promotes T cell–B cell collaboration. Anti‐CD81 induces T cell–B cell adhesion of peripheral blood lymphocytes which is partially mediated by LFA‐1. CD81 engagement promotes LFA‐1‐dependent T cell activation, IL‐2 production and proliferation. The antibody 5A6 was uniquely potent in exerting these effects compared to another antibody to CD81 or to antibodies that react with other tetraspanins expressed on T cells, anti‐CD53 or anti‐CD82. CD81‐derived signals rapidly induce high‐avidity LFA‐1 as measured by cell binding to recombinant ICAM‐3‐coated fluorescent microspheres or by cell adhesion to ICAM‐3‐coated plastic. 5A6 activation of LFA‐1 does not expose the high‐affinity conformation epitope recognized by monoclonal antibody 24.

Differential Expression of Murine CD81 Highlighted by New Anti-Mouse CD81 Monoclonal Antibodies

We describe the use of a soluble CD81-Fc fusion protein to screen for novel monoclonal antibody (MAb) reactive with the extracellular loops of murine CD81 (TAPA-1). Two such MAbs, Eat1 and Eat2 (for Extracellular Anti-TAPA1), were used to assess the expression and function of CD81 on murine lymphocytes. Although CD81 is expressed uniformly on all human lymphocytes, murine CD81 was found to be expressed at much higher levels on resting B cells than on resting T cells. This was particularly evident when staining with the new MAbs, Eat1 and Eat2. The molecule is also functionally active on B cells, as Eat1 and Eat2 induce homotypic adhesion of B lymphocytes. Stimulated B cells undergo early apoptotic events in the presence of Eat2, as shown by binding of Annexin V-fluorescein isothiocyanate (FITC). Polyclonal activation of murine T cells also induces higher level CD81 expression, and many immortalized murine T-cell lines express high levels of the protein. In contrast to human CD81, which is expressed equally on all thymocytes, murine CD81 is induced during thymic development, being expressed at high levels on CD4+CD8+ thymocytes, in contrast to other subsets of thymocytes. Finally, murine dendritic cells, splenic macrophages, and non-killer (NK) cells all express high levels of CD81. We conclude that CD81 is differentially expressed in the murine immune system, and is involved in regulating the adhesion and activation of murine B cells.