Human thrombospondin (TSP) is a high molecular weight glycoprotein composed of three identical disulphide bonded peptide chains. Originally identified as a platelet alpha-granule protein essential for the secondary phase of platelet aggregation, TSP has now been shown to be synthesized by many different cell types including epithelial cells, fibroblasts, smooth muscle cells, endothelial cells and monocytes. In these cells the synthesized TSP is secreted and incorporated into the extracellular matrix (ECM). We have recently shown that TSP is synthesized, secreted and bound to the cell surface by both human keratinocytes and squamous carcinoma cells where it mediates cell-cell and cell-substrate interactions. Further, there is an inverse relationship between the state of differentiation and the ability to synthesize and utilize TSP. Thus, cells that are least differentiated produce the greatest amount of TSP and have the highest number of cell surface receptors for it. We find that there are two classes of receptor for TSP on squamous carcinoma cells that recognize and bind to different regions of the TSP molecule. There is a moderate affinity/high number class of receptor that binds to the heparin binding domain of TSP. This receptor has the characteristics of a heparan sulphate proteoglycan. The other class of receptors is high affinity/low number and binds to the 140 kD fragment of TSP that does not contain the heparin binding domain. This receptor is composed of two cell surface proteins (150/80 kD) and mediates the attachment of squamous carcinoma cells to TSP coated dishes (cell- substrate interaction). In order to better understand the structure and functional role of the 105/80 kD cell adhesion promoting receptor, we propose the following.
Specific Aim 1 : Examine the contribution of the 105/80 kD TSP binding cell surface proteins in cell attachment and cell differentiation. Studies will involve cell attachment and radioligand binding assays, generation of polyclonal and monoclonal antibodies and protein microsequencing of the receptor proteins.
Specific Aim 2 : cDNA cloning of the TSP-Receptor(s). This will be accomplished by screening squamous cell cDNA expression libraries with a combination of antibody and oligonucleotide probes. The cloned cDNA's will be used to study the regulation of the TSP receptor(s) during differentiation.
| Yabkowitz, R; Mansfield, P J; Dixit, V M et al. (1993) Motility of human carcinoma cells in response to thrombospondin: relationship to metastatic potential and thrombospondin structural domains. Cancer Res 53:378-87 |
| Yabkowitz, R; Dixit, V M; Guo, N et al. (1993) Activated T-cell adhesion to thrombospondin is mediated by the alpha 4 beta 1 (VLA-4) and alpha 5 beta 1 (VLA-5) integrins. J Immunol 151:149-58 |
| Yabkowitz, R; Dixit, V M (1991) Human carcinoma cells bind thrombospondin through a Mr 80,000/105,000 receptor. Cancer Res 51:3648-56 |
