The mammalian target of rapamycin (mTOR) regulates cell growth and survival via two different multiprotein complexes, mTORC1 and mTORC2. The assembly of these serine-threonine kinase multiprotein complexes occurs via poorly understood molecular mechanisms. Here, we demonstrate that GRp58/ERp57 regulates the existence and activity of mTORC1. Endogenous mTOR interacts with GRp58/ERp57 in different mammalian cells. In vitro, recombinant GRp58/ERp57 preferentially interacts with mTORC1. GRp58/ERp57 knockdown reduces mTORC1 levels and phosphorylation of 4E-BP1 and p70 S6K in response to insulin. In contrast, GRp58/ERp57 overexpression increases mTORC1 levels and activity. A redox-sensitive mechanism that depends on GRp58/ERp57 expression activates mTORC1. Although GRp58/ERp57 is known as an endoplasmic reticulum (ER) resident, we demonstrate its presence at the cytosol, together with mTOR, Raptor, and Rictor as well as a pool of these proteins associated to the ER. In addition, the presence of GRp58/ERp57 at the ER decreases in response to insulin or leucine. Interestingly, a fraction of p70 S6K, but not 4E-BP1, is associated to the ER and phosphorylated in response to serum, insulin, or leucine. Altogether, our results suggest that GRp58/ERp57 is involved in the assembly of mTORC1 and positively regulates mTORC1 signaling at the cytosol and the cytosolic side of the ER.

G-protein coupled receptors activate heterotrimeric G proteins at the plasma membrane in which most of their effectors are intrinsically located or transiently associated as the external signal is being transduced. This paradigm has been extended to the intracellular compartments by studies in yeast showing that trafficking of G␣ activates phosphatidylinositol 3-kinase (PI3K) at endosomal compartments, suggesting that vesicle trafficking regulates potential actions of G␣ and possibly G␤␥ at the level of endosomes. Here, we show that G␤␥ interacts with Rab11a and that the two proteins colocalize at early and recycling endosomes in response to activation of lysophosphatidic acid (LPA) receptors. This agonist-dependent association of G␤␥ to Rab11a-positive endosomes contributes to the recruitment of PI3K and phosphorylation of AKT at this intracellular compartment. These events are sensitive to the expression of a dominant-negative Rab11a mutant or treatment with wortmannin, suggesting that Rab11a-dependent G␤␥ trafficking promotes the activation of the PI3K/AKT signaling pathway associated with endosomal compartments. In addition, RNA interference-mediated Rab11a depletion, or expression of a dominant-negative Rab11a mutant attenuated LPA-dependent cell survival and proliferation, suggesting that endosomal activation of the PI3K/AKT signaling pathway in response to G␤␥ trafficking, via its interaction with Rab11, is a relevant step in the mechanism controlling these fundamental events.

The calcium-sensing receptor (CaR) helps to maintain the homeostasis of extracellular calcium by controlling the secretion of hormones associated with this process. The mechanism of agonist-induced endocytosis and down-regulation of CaR and the influence of this event on the secretion of CaR-regulated hormones is not fully understood. In this study, we show that CaR is constitutively endocytosed and recycled to the plasma membrane by a Rab11a-dependent mechanism; during this process, the level of total cellular CaR is maintained. This trafficking of CaR promotes the secretion of PTH-related peptide (PTHrP), as evidenced by a decrease on PTHrP secretion in the presence of a dominant-negative mutant of Rab11a. Interestingly, this Rab11a dominant-negative mutant does not interfere with CaR-dependent activation of ERK 1/2, suggesting that ERK signaling is not sufficient to promote PTHrP secretion downstream of CaR. In addition, AMSH (associated molecule with the SH3 domain of STAM), a CaR carboxyl-terminal binding protein, redirects CaR from slow recycling to down-regulation, reducing CaR expression and decreasing PTHrP secretion. Our results indicate that endocytosis and trafficking of CaR modulate PTHrP secretion.

Calcium sensing receptor, a pleiotropic G protein coupled receptor, activates secretory pathways in cancer cells and putatively exacerbates their metastatic behavior. Here, we show that various CaSR mutants, identified in breast cancer patients, differ in their ability to stimulate Rac, a small Rho GTPase linked to cytoskeletal reorganization and cell protrusion, but are similarly active on the mitogenic ERK pathway. To investigate how CaSR activates Rac and drives cell migration, we used invasive MDA‐MB‐231 breast cancer cells. We revealed, by pharmacological and knockdown strategies, that CaSR activates Rac and cell migration via the Gβγ‐PI3K‐mTORC2 pathway. These findings further support current efforts to validate CaSR as a relevant therapeutic target in metastatic cancer.