Plasma membrane amino acid transport is an important site for metabolic control. Most of the regulation for the neutral amino acids is associated with Na+-dependent System A (SysA) activity. Hepatic SysA is stimulated during the cell cycle, by transformation, by amino acid deprivation, and by a variety of cytokines, growth factors, and hormones. The overall hypothesis is that this highly regulated transporter is critical for cell growth and response to the hormonal and nutritional environment. The SysA transporter protein has not been identified nor has a cDNA clone been isolated. The proposed research has two specific aims. First, identification of the glucagon-induced SysA transporter protein, antibody production, and cDNA cloning will be achieved by a multi-faceted approach. Advantage will be taken of the fact that SysA can be immunoprecipitated by antibody against either of the cytoskeletal proteins fodrin or ankyrin. This co-precipitation, in combination with glucagon induction, surface biotinylation, and 35S-methionine-labeling, should yield a finite number of candidate proteins for the preparation of antibodies and subsequent cDNA library screening. If cDNA clones are obtained before the transporter protein is identified, deduced amino acid sequences will be used to prepare antibodies. Confirmation of the cDNA identity will involve characterization of transport in transfected cells and Northern analysis to document the regulation of the mRNA in response to known stimuli of the hepatic SysA transporter. The second specific aim is to use the SysA antiserum to study transporter interaction with ankyrin and fodrin. The working hypothesis is that the ankyrin/fodrin/SysA complexes are critical to the localization and/or regulation of SysA. Plasma membrane domain localization of these complexes will be characterized in control and glucagon-treated rat liver. Measurement of the ankyrin/fodrin/SysA complexes in Golgi subfractions and plasma membrane at specific times following glucagon treatment will establish the localization and kinetics of complex formation.
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