guanosine-diphosphate and Tuberous-Sclerosis

Rheb is a small GTP-binding protein and its GTPase activity is activated by the complex of Tsc1 and Tsc2 whose mutations cause tuberous sclerosis complex (TSC). We previously reported that cultured TSC neurons showed impaired spine synapse morphogenesis in an mTORC1-independent manner. Here we show that the PDZ protein syntenin preferentially binds to the GDP-bound form of Rheb. The levels of syntenin are significantly higher in TSC neurons than in wild-type neurons because the Rheb-GDP-syntenin complex is prone to proteasomal degradation. Accumulated syntenin in TSC neurons disrupts spine synapse formation through inhibition of the association between syndecan-2 and calcium/calmodulin-dependent serine protein kinase. Instead, syntenin enhances excitatory shaft synapse formation on dendrites by interacting with ephrinB3. Downregulation of syntenin in TSC neurons restores both spine and shaft synapse densities. These findings suggest that Rheb-syntenin signalling may be a novel therapeutic target for abnormalities in spine and shaft synapses in TSC neurons.

Topics: Animals; Brain; Chlorocebus aethiops; COS Cells; Dendritic Spines; Ephrin-B3; Guanosine Diphosphate; HEK293 Cells; Hippocampus; Humans; Immunoprecipitation; Mice; Mice, Knockout; Microscopy, Confocal; Monomeric GTP-Binding Proteins; Neurons; Neuropeptides; Patch-Clamp Techniques; Ras Homolog Enriched in Brain Protein; Rats; Synapses; Syntenins; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Rheb (Ras homologue enriched in brain) is a small GTPase that plays an important role in tuberous sclerosis. Here we present the backbone assignments of rRheb in its GDP-bound state. These assignments now provide a basis for the analysis of the interaction of rRheb with putative factors in order to elucidate the function of this GTPase and its role in the MAP kinase pathway of neuronal cells and in tuberous sclerosis.

Topics: Guanosine Diphosphate; Humans; In Vitro Techniques; MAP Kinase Signaling System; Molecular Structure; Monomeric GTP-Binding Proteins; Neuropeptides; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Ras Homolog Enriched in Brain Protein; Recombinant Proteins; Tuberous Sclerosis

The small GTPase Rheb displays unique biological and biochemical properties different from other small GTPases and functions as an important mediator between the tumor suppressor proteins TSC1 and TSC2 and the mammalian target of rapamycin to stimulate cell growth. We report here the three-dimensional structures of human Rheb in complexes with GDP, GTP, and GppNHp (5'-(beta,gamma-imide)triphosphate), which reveal novel structural features of Rheb and provide a molecular basis for its distinct properties. During GTP/GDP cycling, switch I of Rheb undergoes conformational change while switch II maintains a stable, unusually extended conformation, which is substantially different from the alpha-helical conformation seen in other small GTPases. The unique switch II conformation results in a displacement of Gln64 (equivalent to the catalytic Gln61 of Ras), making it incapable of participating in GTP hydrolysis and thus accounting for the low intrinsic GTPase activity of Rheb. This rearrangement also creates space to accommodate the side chain of Arg15, avoiding its steric hindrance with the catalytic residue and explaining its noninvolvement in GTP hydrolysis. Unlike Ras, the phosphate moiety of GTP in Rheb is shielded by the conserved Tyr35 of switch I, leading to the closure of the GTP-binding site, which appears to prohibit the insertion of a potential arginine finger from its GTPase-activating protein. Taking the genetic, biochemical, biological, and structural data together, we propose that Rheb forms a new group of the Ras/Rap subfamily and uses a novel GTP hydrolysis mechanism that utilizes Asn1643 of the tuberous sclerosis complex 2 GTPase-activating protein domain instead of Gln64 of Rheb as the catalytic residue.

Topics: Amino Acid Sequence; Arginine; Asparagine; Binding Sites; Catalytic Domain; Cell Proliferation; Crystallography, X-Ray; Databases, Protein; GTP Phosphohydrolases; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Hydrolysis; Magnesium; Models, Molecular; Molecular Sequence Data; Monomeric GTP-Binding Proteins; Neuropeptides; Protein Conformation; Protein Kinases; Protein Structure, Secondary; Protein Structure, Tertiary; Ras Homolog Enriched in Brain Protein; ras Proteins; Repressor Proteins; Sequence Homology, Amino Acid; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins; X-Ray Diffraction