Depletion of Ric-8B leads to reduced mTORC2 activity

Author summary Gene inactivation in mice can be used to identify genes that are involved in important biological processes and that may contribute to disease. We used this approach to study the Ric-8B gene, which is highly conserved in mammals, including humans. We found that Ric-8B is essential for embryogenesis and for the proper development of the nervous system. Ric-8B mutant mouse embryos are smaller than their wild type littermates and show neural tube defects at the cranial region. This approach also allowed us to identify the biological pathways that potentially contribute to the observed phenotypes, and uncover a novel role for Ric-8B in the mTORC2 signaling pathway. mTORC2 plays particular important roles in the adult brain, and has been implicated in neurological disorders. Our mutant mice provide a model to study the complex molecular and cellular processes underlying the interplay between Ric-8B and mTORC2 in neuronal function. mTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric G alpha s/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway. Phosphorylation of Akt at Ser473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. Knockdown of the endogenous Ric-8B gene in cultured cell lines leads to reduced phosphorylation levels of Akt (Ser473), further supporting the involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2. (AU)