This project seeks to explore the function of a newly discovered protein, TIMAP, in the development of renal glomerular capillaries and the vasculature in general. We have reported evidence in vitro and in vivo, that TGF-Beta1 signals are necessary for renal glomerular capillary morphogenesis. We now extend our observations to the characterization of a novel TGF-Beta1 responsive gene, and its predicted protein product, which we named TIMAP (TGF-Beta1 inhibited membrane associated protein). In vitro, TIMAP is expressed in endothelial and hematopoietic cells (endothelial and hematopoietic cells are derived from a common lineage), and in vivo TIMAP is expressed predominantly in the vasculature and in renal glomeruli, and TIMAP localizes to endothelial cells and VSMC. We find that TIMAP is the first clearly identified direct intracellular binding partner for the non-integrin 67 kDa laminin receptor (67LR) at the cell membrane. The 67LR is a transmembrane glycoprotein, which interacts specifically and with high affinity with the laminin Beta1 chain. 67LR is a marker for highly motile, metastatic cells in human tumors. In endothelial cells, laminin is required for assembly tubes, and inhibition of the interaction between 67LR and laminin blocks endothelial cell organization into tubes. The 67LR is also required for the upregulation of eNOS in endothelial cells in response to shear stress. To date, the mechanisms whereby signals from the 67LR are transmitted to the cell have not been elucidated. We also find that TIMAP interferes with TGF-Beta1 -stimulated endothelial cell apoptosis, apoptosis being component of TGF-Beta1 stimulated glomerular capillary morphogenesis in vitro. Furthermore, in glomerular capillaries in vivo, TGF-Beta1 is involved in removing superfluous cells through the process of apoptosis, and thereby facilitates capillary lumen formation. The current proposal explores the possibility that TIMAP represents a critical component of a 67LR signaling pathway, and thereby is involved in regulating endothelial cell motility, tube formation, and apoptosis.
