VAMP-associated Proteins (VAP) as Receptors That Couple Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Proteostasis with Lipid Homeostasis.

Unesterified cholesterol accumulates in late endosomes in cells expressing the misfolded cystic fibrosis transmembrane conductance regulator (CFTR). CFTR misfolding in the endoplasmic reticulum (ER) or general activation of ER stress led to dynein-mediated clustering of cholesterol-loaded late endosomes at the Golgi region, a process regulated by ER-localized VAMP-associated proteins (VAPs). We hypothesized that VAPs serve as intracellular receptors that couple lipid homeostasis through interactions with two phenylalanines in an acidic track (FFAT) binding signals (found in lipid sorting and sensing proteins, LSS) with proteostasis regulation. VAPB inhibited the degradation of ΔF508-CFTR. The activity was mapped to the ligand-binding major sperm protein (MSP) domain, which was sufficient in regulating CFTR biogenesis. We identified mutations in an unstructured loop within the MSP that uncoupled VAPB-regulated CFTR biogenesis from basic interactions with FFAT. Using this information, we defined functional and physical interactions between VAPB and proteostasis regulators (ligands), including the unfolded protein response sensor ATF6 and the ER degradation cluster that included FAF1, VCP, BAP31, and Derlin-1. VAPB inhibited the degradation of ΔF508-CFTR in the ER through interactions with the RMA1-Derlin-BAP31-VCP pathway. Analysis of pseudoligands containing tandem FFAT signals supports a competitive model for VAP interactions that direct CFTR biogenesis. The results suggest a model in which VAP-ligand binding couples proteostasis and lipid homeostasis leading to observed phenotypes of lipid abnormalities in protein folding diseases.

A cascade of ER exit site assembly that is regulated by p125A and lipid signals.

The inner and outer layers of COPII mediate cargo sorting and vesicle biogenesis. Sec16A and p125A (officially known as SEC23IP) proteins interact with both layers to control coat activity, yet the steps directing functional assembly at ER exit sites (ERES) remain undefined. By using temperature blocks, we find that Sec16A is spatially segregated from p125A-COPII-coated ERES prior to ER exit at a step that required p125A. p125A used lipid signals to control ERES assembly. Within p125A, we defined a C-terminal DDHD domain found in phospholipases and PI transfer proteins that recognized PA and phosphatidylinositol phosphates in vitro and was targeted to PI4P-rich membranes in cells. A conserved central SAM domain promoted self-assembly and selective lipid recognition by the DDHD domain. A basic cluster and a hydrophobic interface in the DDHD and SAM domains, respectively, were required for p125A-mediated functional ERES assembly. Lipid recognition by the SAM-DDHD module was used to stabilize membrane association and regulate the spatial segregation of COPII from Sec16A, nucleating the coat at ERES for ER exit.

AT2 receptors cross talk with AT1 receptors through a nitric oxide- and RhoA-dependent mechanism resulting in decreased phospholipase D activity.

ANG II activation of phospholipase D (PLD) is required for ERK and NAD(P)H oxidase activation, both of which are involved in hypertension. Previous findings demonstrate that ANG II stimulates PLD activity through AT(1) receptors in a RhoA-dependent mechanism. Additionally, endogenous AT(2) receptors in preglomerular smooth muscle cells attenuate ANG II-mediated PLD activity. In the present study, we examined the signal transduction mechanisms used by endogenous AT(2) receptors to modulate ANG II-induced PLD activity through either PLA(2) generation of lysophosphatidylethanolamine or Galpha(i)-mediated generation of nitric oxide (NO) and interaction with RhoA. Blockade of AT(2) receptors, Galpha(i) and NO synthase, but not PLA(2), enhanced ANG II-mediated PLD activity in cells rich in, but not poor in, AT(2) receptors. Moreover, NO donors, a direct activator of guanylyl cyclase and a cGMP analog, but not lysophosphatidylethanolamine, inhibited ANG II-mediated PLD activity, whereas an inhibitor of guanylyl cyclase augmented ANG II-induced PLD activity. AT(2) receptor- and NO-mediated attenuation of ANG II-induced PLD activity was completely lost in cells transfected with S188A RhoA, which cannot be phosphorylated on serine 188. Therefore, our data indicate that AT(2) receptors activate Galpha(i), subsequently stimulating NO synthase and leading to increased soluble guanylyl cyclase activity, generation of cGMP, and activation of a protein kinase, resulting in phosphorylation of RhoA on serine 188. Furthermore, because AT(2) receptors inhibit AT(1) receptor signaling to PLD via modulating RhoA activity, AT(2) receptor signaling can potentially regulate multiple vasoconstrictive signaling systems through inactivating RhoA.

The guanine nucleotide exchange factor ARNO mediates the activation of ARF and phospholipase D by insulin.

BACKGROUND: Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. RESULTS: Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: DeltaCC-ARNO and CC-ARNO were partially translocated to the membranes while DeltaPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. CONCLUSIONS: Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor.

Activation of phospholipase D by the small GTPase Sar1p is required to support COPII assembly and ER export.

The small GTPase Sar1p controls the assembly of the cytosolic COPII coat that mediates export from the endoplasmic reticulum (ER). Here we demonstrate that phospholipase D (PLD) activation is required to support COPII-mediated ER export. PLD activity by itself does not lead to the recruitment of COPII to the membranes or ER export. However, PLD activity is required to support Sar1p-dependent membrane tubulation, the subsequent Sar1p-dependent recruitment of Sec23/24 and Sec13/31 COPII complexes to ER export sites and ER export. Sar1p recruitment to the membrane is PLD independent, yet activation of Sar1p is required to stimulate PLD activity on ER membranes, thus PLD is temporally regulated to support ER export. Regulated modification of membrane lipid composition is required to support the cooperative interactions that enable selective transport, as we demonstrate here for the mammalian COPII coat.

The role of phosphatidic acid in the regulation of the Ras/MEK/Erk signaling cascade.

Phosphatidic acid (PA) is an important second messenger produced by the activation of numerous cell surface receptors. Recent data have suggested that PA regulates multiple cellular processes. This review addresses primarily the role of PA in the regulation of the Erk1/2 cascade pathway. A model for the regulation of Erk1/2 phosphorylation by cell surface receptors is presented. According to this model, agonists stimulate the binding of GTP to Ras and the activation of phospholipase D to generate phosphatidic acid. PA promotes the binding of cRaf-1 kinase to the membrane, where it interacts with Ras.GTP and other regulatory components of the pathway. Ras-Raf complexes remain bound to the surface of endosomes, where scaffolding complexes involving Ras, cRaf-1, MEK and Erk are formed. Complete activation and coupling of the cascade requires endocytosis, a process that is also modulated by PA.