Membrane proteins are divided into two groups based upon the ease with which the proteins can be removed from the membrane. Extrinsic or peripheral membrane proteins can be removed using extremes of ionic strength or pH, the use of urea or other disruptors of protein interactions. Intrinsic or integral membrane proteins are released only when the lipid bilayer of the membrane is dissolved by detergent.
The majority of known integral membrane proteins are transmembrane proteins which comprises an extracellular, a transmembrane, and an intracellular domain. Transmembrane proteins are typically embedded into the cell membrane by one or more regions comprising 15 to 25 hydrophobic amino acids which are predicted to adopt an .alpha.-helical conformation. Transmembrane proteins are classified as bitopic (or Types I and II) and polytopic (or Types III and IV) Singer, S. J. (1990) Annu. Rev. Cell Biol. 6:247-96!. Bitopic proteins span the membrane once while polytopic proteins contain multiple membrane-spanning segments. Type III integral membrane proteins have multiple transmembrane stretches of hydrophobic residues. Transmembrane proteins carry out a variety of important cellular functions such as acting as cell-surface receptor proteins which are involved in signal transduction (e.g., growth factor receptors) and transport of ions or metabolites (e.g., ion channels).
Recently a multigene family encoding type III integral membrane proteins, termed the transmembrane 4 superfamily (TM4SF) or tetraspan family, was identified Wright, M. D. and Tomlinson, M. G. (1994) Immunol. Today 15:588!. The TM4SF family comprises a superfamily of membrane proteins which cross or traverse the cell membrane four times. Members of the TM4SF include a number of platelet and endothelial cell membrane proteins PETA-3, CD9 (lung adenocarcinoma antigen MRP-1), the platelet and melanoma-associated antigen CD63 (ME491)!, leukocyte surface glycoproteins CD53, CD37, CD63, R2, and the tumor associated antigen TAPA-1 (CD81)!, the colonal carcinoma antigen CO-029, mink lung epithelial protein TI-1, the tumor-associated antigen L6, SAS (a gene amplified in human sarcomas), the product of a gene responsible for slow retinal degeneration in mice (the rds gene product), and surface proteins of the schistosome parasites Fitter, S. et al. (1995) Blood 86:1348; Boucheix et al. (1991) J. Biol. Chem. 266:117; Wright, M. D. et al. (1993), Int. Immunol. 5:209; Classon, B. J. (1989) J. Exp. Med. 169:1497 and (1990) J. Exp. Med. 172:1007; Hotta, J. et al. (1988) Cancer Res. 48:2955; Gaugitsch, T. et al. (1991) Eur. J. Immunol. 21:377; Oren, R. et al. (1990) Mol. Cell. Biol. 10:4007; Szala, S. et al. Proc. Natl. Acad. Sci. USA 87:6833; Kallin, B. et al. (1991) Mol. Cell. Biol. 11:5338; Miyake, M. (1991) J. Exp. Med. 174:1347; Marken, J. S. et al. (1992) Proc. Natl. Acad. Sci. USA 89:3503; Travis, G. H. et al. (1991) Genomics 10:733-739, Jankowski, S. A. (1994) Oncogene 9:1205; and Wright, M. D. et al. (1990) J. Immunol. 144:3195!. The members of the TM4SF show about 25-30% amino acid sequence identity with one another.
The predicted structure of the TM4SF proteins reveals a topology where the N- and C-termini are intracellular and the major hydrophilic domain, located between transmembrane domains 3 and 4, is extracellular. TM4SF members are most conserved in their transmembrane and cytoplasmic domains (the conservation among transmembrane domains being the highest) and most divergent in their two hydrophilic extracellular domains. The high level of conservation seen in the transmembrane and cytoplasmic domains of TM4SF members suggest an effector/signaling function common to all members. The divergence of the extracellular domains suggests these domains provide functions specific to each family member such as ligand binding or protein-protein interaction Wright, M. D. et al. (1993), supra and Fitter, S. et al., supra!.
A number of TM4SF members have been implicated in signal transduction, control of cell adhesion, regulation of cell growth and proliferation (including development and oncogenesis) and motility (including the ability to suppress metastatic potential) and expression of a number of TM4SF members is associated with a variety of tumors (e.g., CD81/TAPA-1,L6, CD9/MRP-1, L6, CD63/ME491, CO-029, SAS, PETA-3). Indeed the expression of several TM4SF members is altered when cells are growing or activated. CD9, CD53 and CD82 are upregulated when lymphocytes are activated. Cell surface expression of CD37 is rapidly lost upon activation of B cells. Other TM4SF members are implicated in cell growth due to their association with tumor cells. CD9 (MRP-1) is a marker for 90% of non-T acute lymphoblastic leukemia cells and 50% of acute myeloid and chronic lymphoid leukemias; CD9 is not expressed on resting B and T lymphocytes. Anti-CD9 antibodies inhibits the motility of a variety of cancer cell lines and inhibits the metastatic potential of the mouse BL6 cell line, a highly metastatic variant of B16 cells Miyake, M. and Hakomori, S. (1991) Biochem. 30:3328!. Expression of CD9 in transfection experiments correlated with suppression of metastatic potential and cell motility Ikeyama, S. et al. (1993) J. Exp. Med. 174:1347!. CD63 (ME491) is expressed in early stage melanoma but is downregulated in advanced stages of melanoma; CD63 is not expressed on normal tissue melanocytes. CO-029 is expressed on colon, gastric, pancreatic and rectal carcinomas but not on most normal tissues. The gene encoding SAS is amplified in a subset of human sarcomas.
CD53 is a recently characterized member of the TM4SF Amiot, M. (1990) J. Immunol. 145:4322 and Wright, M. D. et al. (1993), supra! (CD53 is also known as the rat OX44 antigen). CD53 is a glycoprotein which is expressed on the surface of all nucleated cells of hematopoietic origin and CD53 transcripts are dramatically increased following cell activation. Among the TM4SF members, CD53 is most closely related to CD37 and CD82 (R2), a marker which is upregulated upon activation of B and T cells (Wright, M. D. and Tomlinson, M. G., supra). CD53 is distantly related to a bacterial type III integral membrane protein that transports lactose into cells (E. coli lac Y permease) and it has been suggested in view of this homology and the upregulation of CD53 following mitogenic stimulation of lymphocytes that CD53 may be involved in the transport of factors required for cell proliferation Amiot, M., supra!. CD53, like other members of the TM4SF, has been shown to be a component of transmembrane signaling complexes. CD53 associates with CD2 on T cells and natural killer cells Bell, G. M. (1992) J. Exp. Med. 175:527! and it is found in a complex containing CD37, TAPA-1, CD82 and MHC class I or II molecules in B cells Angelisova, P. (1994) Immunogenetics 39:249!.
Biochemical data demonstrates that CD53 is coupled to signal transduction pathways. Anti-CD53 antibodies cause calcium fluxes in human B cells, granulocytes and monocytes along with activation of the monocyte oxidative burst Olweus, J. et al. (1993) J. Immunol. 151:707!. In the rat, anti-CD53 antibodies stimulate an inositol phosphate response, protein phosphorylation and an increase in intracellular free calcium; the anti-rat CD53 antibody 7D2 is mitogenic for splenic T cells Bell, G. M. et al., supra!. Thus, CD53 is involved in the regulation of cell proliferation and activation.
The discovery of molecules related in the TM4SF multigene family satisfies a need in the art by providing new diagnostic or therapeutic compositions useful in the treatment of disorders associated with alterations in the expression of members of the TM4SF multigene family.