Molecular Mechanisms Of Rag GTPases Protein Complex Mediated Amino Acid Activation Of MTORC1

How cell growth is regulated represents one of the fundamental issues in biology. Studies over the last two decades established TOR(Target of Rapamycin) as a master regulator of cell growth. TOR signaling pathway is highly conserved from yeast to mammals. mTORC1(Mammalian Target of Rapamycin complex1) signaling pathway integrates a variety of environmental cues to regulate cell growth. Growth factors, Energy status and amino acids are the major upstream signal inputs for mTORC1activation and the presence of all three signal inputs is necessary for mTORC1activation. Activated mTORC1positively regulates a variety of major biological processes including protein synthesis, lipid synthesis and nucleotide synthesis, thereby promoting cell growth.The signaling cascades mediating growth factors or energy status to mTORC1activation have been well defined. However, how amino acids activate mTORCl remains elusive until recently. In2008, two groups independently discovered that Rag GTPases mediated amino acid signal to mTORC1.Rag GTPases are a group of small GTPases that belongs to Ras GTPase family. There are four members in human:RagA, RagB, RagC and RagD. RagA and RagB are highly similar to each other while RagC and RagD are highly similar to each other. RagA or RagB forms an obligatory heterodimer with RagC or RagD. To our knowledge, this is the first GTPase heterodimer in Ras GTPase family. Rag GTPases are highly conserved from yeast to mammals. Gtrlp is the yeast ortholog of RagA and RagB whereas Gtr2p is the yeast ortholog of RagC and RagD. Similar to Rag GTPases, Gtrlp and Gtr2p also form a heterodimer.Rag GTPases is anchored to lysosome through its interaction with Ragulator protein complex. Another small GTPase Rheb, which is responsible for direct mTORC1activation, also resides on lysosome and its activation requires growth factor and energy inputs. When cells amino acid is abundant, Rag GTPases are activated. Rag GTPases are fully activated when RagA/B is loaded with GTP while RagC/D is loaded with GDP. Activated Rag GTPases interacts with mTORC1component Raptor and recruits mTORC1to the lysosomal surface for activation by lysosomal Rheb.Although the role of Rag GTPases in relaying amino acid singal to mTORC1has been established, the molecular mechanisms by which Rag GTPases interacts with and recruits mTORC1to the lysosome remain poorly understood. To understand the mechanism of Rag GTPases mediated TORC1activaiton, we solved the crystal structure of Gtrlp-Gtr2p, the yeast orthologs of Rag GTPases, bound to GMPPNP at2.8A. The overall structure of Gtrlp-Gtr2p adopts pseudo-two fold symmetry and resembles a U shaped horseshoe. The structure of Gtrlp and Gtr2p are highly similar. The GTPase domains of Gtrlp and Gtr2p are at the same side of the C terminal dimer and close to each other, with the switch regions facing at opposite direction. However, no direct interactions were observed. Gtrlp and Gtr2p form a heterodimer via extensive interactions with their C terminal dimerization domain. Since the biological functions of Rag GTPases are highly conserved from yeast to mammals and human mTORC1is more extensively studied, we used the human RagA-RagC complex to investigate their function in TORC1binding and activation, guided by structural analysis of Gtrlp-Gtr2p. First, we found that both the GTPase domains and C terminal dimerization domains of Rag GTPases are necessary for amino acid induced mTORC1activation. Second, we found that the switch region of RagA is critical for Raptor binding and amino acid indcued mTORC1activation. Third, the C terminal dimer of RagA-RagC is necessary and sufficient to interact with the Ragulator component P18, which enables Rag GTPases to recruit mTORC1to the lysosomal surface upon amino acid stimulation. Interestly, the structure of the C terminal dimer of RagA-RagC is highly similar to that of the Ragulator components MP1-P14. However, the biological significance of this similarity is unclear. In total, this study revealed the molecular mechanism by which Rag GTPases mediates amino acid induced mTORC1activation. Since mTORC1activity is highly related to cancer and aging, this study also provides new insights into therapeutic targeting of mTORC1for cancer treatment and longevity extention.