Organic anions such as the bile acids play a critical role in numerous physicological processes such as digestion of dietary lipids and regulation of cholesterol levels. These compounds are transported into the liver by a 49 kDa protein located in the sinusoidal plasma membrane. Defects in this transport system can result in intrahepatic cholestasis leading to numerous pathological conditions including liver injury and failure. The purpose of this research program is to investigate the structure of this transport system and its expression in different physiological and pathological states.
The specific aims of the proposal are: 1) to isolate a cDNA clone for the Na+ -dependent anion transport protein. The amino acid sequence of this protein will be obtained by sequencing this cDNA clone; 2) to characterize the membrane organization as well as functionally important sites on this transport protein using a library of monoclonal antibodies, protein site specific and membrane domain specific labeling reagents and proteolytic dissection in conjunction with the sequences data obtained in (1); 3) to investigate the structure of a 49 kDa non- functioning transport protein in hepatoma cell plasma membranes; 4) to utilize the cDNA probe of the anion transport protein to characterize the steady state levels of mRNA for this protein in liver in different stages of development, during regeneration, in transformed hepatocytes and in other tissues; 5) to characterize the expression of this transport protein in the hepatocyte system described in (4) using mono and polyclonal antibodies against the transport protein; 6) to transfect the cDNA clone for this protein into non-transporting heptoma cells. These studies, which will provide important new information on the structure of the Na+ - dependent organic anion transport system and its expression in different physiological states, should ultimately lead to an understanding of the mechanism of action and regulation of this membrane system.
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