The mechanism of translational control of the arginine/lysine transporter mRNA by amino acid availability will be studied. The essential amino acids arginine and lysine, are mainly transported via the cat-1 (cationic amino acid transporter 1) protein of the Y+ system. The cat-1 gene is expressed in all cell types and in high levels in proliferating cells, emphasizing the importance of the cat-1 protein for growth and development of mammals. Mammalian cells have developed an adaptive response to changes in amino acid availability. When the amino acid supply is limited, protein synthesis decreases and there are increases in catabolism of cellular proteins, amino acid biosynthesis, and amino acid transport across the plasma membrane. Together these responses provide the amino acids, which are essential for cell survival. A significant part of this adaptive response is the increased expression of the cat-1 gene. This involves the coordinate increases in transcription, mRNA stability and translation, thus enabling the cells to transport the essential amino acids lysine and arginine once they become available. This proposal will study a novel mechanism, used for the synthesis of the cat-1 protein during limited amino acid supply, when global protein synthesis is inhibited. This mechanism involves translation initiation via an internal ribosomal entry sequence (IRES), a mechanism known to regulate translation of viral mRNAs in infected cells. This IRES is found within the 5'-untranslated region of the cat-1 mRNA. The studies proposed will determine the molecular events that lead to increased IRES-mediated translation of the transporter mRNA during amino acid starvation. Our hypothesis is that during amino acid starvation a protein is synthesized which by binding within the 5'-untranslated region of the cat-1 mRNA, mediates an active IRES conformation. We will determine the following: (1) the cell signaling pathway leading to IRES-activation (2) the cis-mRNA sequence/structure for IRES- activity (3) the trans-acting factors that mediate IRES- conformational change and translational activity. Delineation of the molecular mechanisms of regulation of essential amino acid transport into mammalian cells by substrate availability, will be a valuable guide of how to improve human health during catabolic stress conditions.
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