Regulation of amino acid-sensitive TOR signaling by leucine analogues in adipocytes

In adipocytes, amino acids stimulate the target of rapamycin (TOR) signaling pathway leading to phosphorylation of the translational repressor, elF-4E binding protein-I (4E-BP1), and ribosomal protein S6. L-leucine is the primary mediator of these effects. The structure-activity relationships of a putative L-leucine recognition site in adipocytes (LeuR(A)) that regulates TOR activity were analyzed by examining the effects of leucine analogues on the rapamycin-sensitive phosphorylation of the translational repressor, elF- 4E binding protein-I (4E-BP1), an index of TOR activity. Several amino acids that are structurally related to leucine strongly stimulated 4E-BP1 phosphorylation at concentrations greater than the EC₅₀ value for leucine. The order of potency was leucine > norleucine > threo-L-β-hydioxyleucine ≃ lle > Met ≃ Val. Other structural analogues of leucine, such as H-α-methyl- D/L-leucine, S-(-)-2-amino-4-pentenoic acid, and 3-amino-4-methylpentanoic acid, possessed only weak agonist activity. However, other leucine-related compounds that are known agonists, antagonists, or ligands of other leucine binding/recognition sites did not affect 4E-BP1 phosphorylation. We conclude from the data that small lipophilic modifications of the leucine R group and α-hydrogen may be tolerated for agonist activity; however, leucine analogues with a modified amino group, a modified carboxylic group, charged R groups, or bulkier aliphatic R groups do not seem to possess significant agonist activity. Furthermore, the leucine recognition site that regulates TOR signaling in adipocytes appears to be different from the following: (1) a leucine receptor that regulates macroautophagy in liver, (2) a leucine recognition site that regulates TOR signaling in H411E hepatocytes, (3) leucyl tRNA or leucyl tRNA synthetase, (4) the gabapentin-sensitive leucine transaminase, or (5) the system L-amino acid transporter. (C) 2000 Wiley- Liss, Inc.