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Lipoproteins: transmembrane signalling and mitogenic potential in vascular smooth muscle cells

A primary objective of our planned research activities was to determine the molecular identity of the "atypical" lipoprotein binding proteins (p105 and p130) which we have shown to be present on smooth muscle cells (SMC). A procedure was developed for the purification of p105 and p130 from human aortic medial tissue. Using a combination of biochemical (partial amino acid sequencing) and immunological (generation of peptide antisera) techniques the "atypical" lipoprotein-binding proteins (Mr 105 and 130) present on smooth muscle cells (SMC) have been identified as T-cadherin, a glycosyl phosphoinositide (GPI)-anchored cell adhesion glycoprotein.

Cadherins are a family of cellular adhesion proteins mediating homotypic cell-cell binding. In contrast to classical cadherins, T-cadherin does not possess the transmembrane and cytosolic domains known to be essential for tight mechanical coupling of cells, and is instead attached to the cell membrane by a glycosylphosphatidylinositol (GPI)-anchor. Thus we explored the hypothesis that T-cadherin might function as a signal-transducing protein. T-cadherin (p105/p130) is enriched in detergent-insoluble membrane domains of SMC (so-called caveolae) within which other GPI-anchored proteins (CD-59, uPA receptor) and signal transducing molecules (Gas and src-kinases) co-localize. The coupling of T-cadherin with such signalling molecules within caveolae might enable T-cadherin mediated signal transduction.

Although cadherins are generally recognized to be important regulators of morphogenesis, the function of T-cadherin in the vasculature is poorly understood. We investigated whether in vitro manipulation of SMC growth characteristics of SMC might influence T-cadherin protein expression levels. T-cadherin protein (p105/p130) levels in SMC are regulated by stimulatory (downmodulation) and inhibitory (upmodulation) growth regulators. Protein levels in SMC cultures are dependent upon cell density and proliferation status, in a manner that suggests T-cadherin may function as a negative determinant of SMC growth.

The complete cDNA coding sequence of human pre pro T-cadherin was generated. cDNA fragments suitable for expression in eukaryotic (CHO, HEK293) and prokaryotic (E.Coli) systems. Eukaryotic cells transfected (transient and stable) with T-cadherin cDNA express two proteins (105 and 130 kDa) which, on Western blots, show LDL binding and immunoreactivity with anti-T-cadherin peptide antisera. Both proteins can be removed from the surface of cultures by PI-PLC (i.e. GPI-anchored with correct outward orientation) and the mature protein (p105) is insensitive to trypsin proteolysis in the presence of calcium (a known property of T-cadherin).

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