No abstract

The atypical low density lipoprotein (LDL) binding proteins (M r 105 and 130 kDa ; p105 and p130) in human aortic medial membranes and cultured human and rat aortic smooth muscle cells (SMC) have recently been identified as the cell adhesion glycoprotein T-cadherin. Although cadherins are generally recognized to be important regulators of morphogenesis, the function of T-cadherin in the vasculature is poorly understood. This study has examined the relationship between expression of T-cadherin and the density and proliferation status of SMC. T-cadherin (p105 and p130) levels in SMC lysates were measured on Western blots using ligand-binding techniques. T-cadherin expression was dependent upon cell density, and maximal levels were achieved at confluency. T-cadherin levels were reversibly modulated by switching cultures between serumfree (upmodulation) and serum-containing (downmodulation) conditions. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor (EGF) or insulin-like growth factor (IGF) elicited a dose-and time-dependent downmodulation that was reversible after transfer of SMC to growth factor-free medium. Our results support the hypothesis that T-cadherin may function as a negative determinant of cell growth.z 1998 Federation of European Biochemical Societies.

By use of ligand-blotting techniques, this study investigated lipoprotein-binding proteins in human aortic smooth muscle. PAGE was performed under non-reducing conditions, and, using low-density lipoprotein (LDL) as ligand, with rabbit anti-apolipoprotein (apo) B and 125I-labelled goat anti-rabbit IgG as primary and secondary antibodies respectively, we demonstrate that membranes from human aortic media (and cultured human smooth-muscle cells) contain a major lipoprotein-binding protein with an apparent molecular mass of 105 kDa. Anionized preparations (carbamoyl- and acetyl-) of LDL, which did not displace 125I-LDL bound to the apo B,E receptor of cultured fibroblasts, were also recognized as ligands for the 105 kDa protein in aortic media membranes. LDL binding to 105 kDa protein was decreased in the presence of high density lipoprotein (HDL), although more than 100-fold molar excess of HDL was required to achieve 50% displacement of bound LDL. The LDL-binding activity of 105 kDa protein was inhibited by EDTA, and was also significantly decreased when samples were reduced by beta-mercaptoethanol before electrophoresis. Monoclonal antibodies against apo B,E receptor reacted with partially purified bovine adrenal apo B,E receptor, but not with 105 kDa protein of human aortic media membranes. The spectrum of properties of this vascular smooth-muscle lipoprotein-binding protein binding are clearly distinct from those of other previously characterized lipoprotein-binding molecules.

T-cadherin (T-cad) is an unusual glycosylphosphatidylinositol-anchored member of the cadherin family of cell adhesion molecules. Binding of low density lipoproteins (LDLs) to T-cad can be demonstrated on Western blots of smooth muscle cell lysates, membranes and purified proteins. Using HEK293 cells transfected with human T-cad cDNA (T-cad+), we have investigated the adhesion properties of expressed mature and precursor proteins and examined the postulate that LDL represents a physiologically relevant ligand for T-cad. T-cad+ exhibits an increased Ca 2+ -dependent aggregation (vs. control) that was reduced by selective proteolytic cleavage of precursor T-cad and abolished after either proteolytic or phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of both mature and precursor proteins, indicating that both proteins function in intercellular adhesion. T-cad+ exhibited a significantly increased specific cell surface-binding of [125 I]-LDL that was sensitive to PI-PLC pre-treatment of cells. Ca 2+ -dependent intercellular adhesion of T-cad+ was significantly inhibited by LDL. Our results support the suggestion that LDL is a physiologically relevant ligand for T-cad.z 1999 Federation of European Biochemical Societies.

Atypical cell surface lipoprotein-binding proteins of 105 kDa and 130 kDa are present in membranes of vascular smooth muscle cells. We recently identified the 105 kDa protein from human aortic media as T-cadherin, an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. The goal of the present study was to determine the identity of 130 kDa lipoprotein-binding protein of smooth muscle cells. We applied different approaches that included protein sequencing of purified protein from human aortic media, the use of human T-cadherin peptide-specific antisera, and enzymatic treatment of cultured cells with trypsin and GPI-specific phospholipase C. Our results indicate that the 130 kDa protein is a partially processed form of T-cadherin which is attached to the membrane surface of smooth muscle cells via a GPI anchor and contains uncleaved N-terminal propeptide sequence. Our data disclose that, in contrast to classical cadherins, T-cadherin is expressed on the cell surface in both its precursor (130 kDa) and mature (105 kDa) forms.