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Richard G W Anderson

Publications and source records attributed to Richard G W Anderson.

At least 19 recordsLinked to original sources
PubMed2007

Lipidomics reveals that adiposomes store ether lipids and mediate phospholipid traffic.

Lipid droplets are accumulations of neutral lipids surrounded by a monolayer of phospholipids and associated proteins. Recent proteomic analysis of isolated droplets suggests that they are part of a dynamic organelle system that is involved in membrane traffic as well as packaging and distributing lipids in the cell. To gain a better insight into the function of droplets, we used a combination of mass spectrometry and NMR spectroscopy to characterize the lipid composition of this compartment. In addition to cholesteryl esters and triacylglycerols with mixed fatty acid composition, we found that approximately 10-20% of the neutral lipids were the ether lipid monoalk(en)yl diacylglycerol. Although lipid droplets contain only 1-2% phospholipids by weight, >160 molecular species were identified and quantified. Phosphatidylcholine (PC) was the most abundant class, followed by phosphatidylethanolamine (PE), phosphatidylinositol, and ether-linked phosphatidylcholine (ePC). Relative to total membrane, droplet phospholipids were enriched in lysoPE, lysoPC, and PC but deficient in sphingomyelin, phosphatidylserine, and phosphatidic acid. These results suggest that droplets play a central role in ether lipid metabolism and intracellular lipid traffic.

Biological Transport↗
PubMed2007

Lipid rafts: at a crossroad between cell biology and physics.

Membrane lateral heterogeneity is accepted as a requirement for the function of biological membranes and the notion of lipid rafts gives specificity to this broad concept. However, the lipid raft field is now at a technical impasse because the physical tools to study biological membranes as a liquid that is ordered in space and time are still being developed. This has lead to a disconnection between the concept of lipid rafts as derived from biochemical and biophysical assays and their existence in the cell. Here, we compare the concept of lipid rafts as it has emerged from the study of synthetic membranes with the reality of lateral heterogeneity in biological membranes. Further application of existing tools and the development of new tools are needed to understand the dynamic heterogeneity of biological membranes.

Biology↗
PubMed2006

An intimate collaboration between peroxisomes and lipid bodies.

Although peroxisomes oxidize lipids, the metabolism of lipid bodies and peroxisomes is thought to be largely uncoupled from one another. In this study, using oleic acid-cultured Saccharomyces cerevisiae as a model system, we provide evidence that lipid bodies and peroxisomes have a close physiological relationship. Peroxisomes adhere stably to lipid bodies, and they can even extend processes into lipid body cores. Biochemical experiments and proteomic analysis of the purified lipid bodies suggest that these processes are limited to enzymes of fatty acid beta oxidation. Peroxisomes that are unable to oxidize fatty acids promote novel structures within lipid bodies ("gnarls"), which may be organized arrays of accumulated free fatty acids. However, gnarls are suppressed, and fatty acids are not accumulated in the absence of peroxisomal membranes. Our results suggest that the extensive physical contact between peroxisomes and lipid bodies promotes the coupling of lipolysis within lipid bodies with peroxisomal fatty acid oxidation.

Acyl-CoA Oxidase↗
PubMed2006

Detergent-free caveolae proteome suggests an interaction with ER and mitochondria.

Recent proteomic studies of detergent resistant membrane fractions have begun to characterize the protein composition of caveolae and lipid rafts. The methods used in most of these studies, however, are not able to distinguish between plasma membrane and internal membrane lipid domains. Here we used a non-detergent method for obtaining fractions enriched in caveolae derived from the plasma membrane of multiple cell types. Unexpectedly, the proteins in the caveolae proteome suggest these lipid domains may interact with elements of ER and mitochondria. A comparison of the partial proteome we obtained with other published reports identifies 26 proteins that are candidate marker proteins for identifying caveolae in multiple cell types.

Caveolae↗
PubMed2005

Phospho-caveolin-1 mediates integrin-regulated membrane domain internalization.

Growth of normal cells is anchorage dependent because signalling through multiple pathways including Erk, phosphatidylinositol-3-OH kinase (PI(3)K) and Rac requires integrin-mediated cell adhesion. Components of these pathways localize to low-density, cholesterol-rich domains in the plasma membrane named 'lipid rafts' or 'cholesterol-enriched membrane microdomains' (CEMM). We previously reported that integrin-mediated adhesion regulates CEMM transport such that cell detachment from the extracellular matrix triggers CEMM internalization and clearance from the plasma membrane. We now report that this internalization is mediated by dynamin-2 and caveolin-1. Internalization requires phosphorylation of caveolin-1 on Tyr 14. A shift in localization of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalization upon cell detachment, which mediates inhibition of Erk, PI(3)K and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1.

Animals↗
PubMed2005

Caveolin-1 is not required for murine intestinal cholesterol transport.

Caveolin-1 (CAV1) is the structural protein of the filamentous coat that decorates the cytoplasmic surface of each caveola. Cell culture studies have implicated CAV1 in playing an important role in intracellular cholesterol trafficking. In addition, it has been reported that CAV1 forms a detergent-resistant protein complex with Annexin-2 in enterocytes that can be disrupted by the cholesterol absorption inhibitor ezetimibe, suggesting a possible role for CAV1 in cholesterol absorption. In this report, we have evaluated cholesterol homeostasis in Cav1 knock-out mice. Deletion of CAV1 does not result in either a compensatory increase of CAV2 or CAV3 in intestine. In addition, Cav1 knock-out mice display normal mRNA and protein levels of Annexin-2 or the putative cholesterol transport protein Niemann-Pick C1-like 1 (NPC1L1) in proximal intestinal mucosa. Fractional cholesterol absorption and fecal neutral sterol excretion are statistically similar in Cav1 knock-out mice and their wild-type littermates. Moreover, oral administration of ezetimibe is equally effective in decreasing cholesterol absorption in Cav1 null mice and wild-type controls. The mRNA expression levels of genes sensitive to intracellular cholesterol concentration (ATP-binding cassette transporters ABCA1 and ABCG5, hydroxymethylglutaryl-CoA synthase and the LDL receptor) are similarly altered in the proximal intestinal mucosa of Cav1 null and wild-type mice following ezetimibe treatment. These results demonstrate that CAV1 is not required for cholesterol absorption or ezetimibe sensitivity in the mouse.

ATP Binding Cassette Transporter 1↗
PubMed2005

Low density lipoprotein receptor-related protein 1 (LRP1) controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor beta (PDGFR beta).

The low density lipoprotein receptor-related protein 1 (LRP1) has been implicated in intracellular signaling functions as well as in lipid metabolism. Recent in vivo and in vitro studies suggest that LRP1 is a physiological modulator of the platelet-derived growth factor (PDGF) signaling pathway. Here we show that in mouse fibroblasts LRP1 modulates PDGF-BB signaling by controlling endocytosis and ligand-induced down-regulation of the PDGF receptor beta (PDGFRbeta). In LRP1-deficient fibroblasts, basal PDGFRbeta tyrosine kinase activity was derepressed, and PDGF-BB-induced endocytosis and degradation of PDGFRbeta were accelerated as compared with control cells. This was accompanied by rapid uptake of receptor-bound PDGF-BB into the cells and by attenuated ERK activation in response to PDGF-BB stimulation. Pulse-chase analysis indicated that the steady-state turnover rate of PDGFRbeta was also accelerated in LRP-deficient fibroblasts. The rapid degradation of PDGFRbeta in the LRP1-deficient fibroblasts was prevented by MG132 and chloroquine. Furthermore, the association of PDGFRbeta with c-Cbl, a ubiquitin E3-ligase, as well as the ligand-induced ubiquitination of PDGFRbeta were increased in LRP1-deficient fibroblasts. We show that LRP1 can directly interact with c-Cbl, suggesting a Sprouty-like role for LRP1 in regulating the access of the PDGFRbeta to the ubiquitination machinery. Thus, LRP1 modulates PDGF signaling by controlling ubiquitination and endocytosis of the PDGFRbeta.

Animals↗
PubMed2005

OSBP is a cholesterol-regulated scaffolding protein in control of ERK 1/2 activation.

Oxysterol-binding protein (OSBP) is the founding member of a family of sterol-binding proteins implicated in vesicle transport, lipid metabolism, and signal transduction. Here, OSBP was found to function as a cholesterol-binding scaffolding protein coordinating the activity of two phosphatases to control the extracellular signal-regulated kinase (ERK) signaling pathway. Cytosolic OSBP formed a approximately 440-kilodalton oligomer with a member of the PTPPBS family of tyrosine phosphatases, the serine/threonine phosphatase PP2A, and cholesterol. This oligomer had dual specific phosphatase activity for phosphorylated ERK (pERK). When cell cholesterol was lowered, the oligomer disassembled and the level of pERK rose. The oligomer also disassembled when exposed to oxysterols. Increasing the amount of OSBP oligomer rendered cells resistant to the effects of cholesterol depletion and decreased the basal level of pERK. Thus, cholesterol functions through its interaction with OSBP outside of membranes to regulate the assembly of an oligomeric phosphatase that controls a key signaling pathway in the cell.

Binding Sites↗
PubMed2005

Signaling networks in living cells.

Recent advances in cell signaling research suggest that multiple sets of signal transducing molecules are preorganized and sequestered in distinct compartments within the cell. These compartments are assembled and maintained by specific cellular machinery. The molecular ecology within a compartment creates an environment that favors the efficient and accurate integration of signaling information arriving from humoral, mechanical, and nutritional sources. The functional organization of these compartments suggests they are the location of signaling networks that naturally organize into hierarchical interconnected sets of molecules through their participation in different classes of interacting units. An important goal is to determine the contribution of the compartment to the function of these networks in living cells.

Animals↗
PubMed2004

The modular adaptor protein ARH is required for low density lipoprotein (LDL) binding and internalization but not for LDL receptor clustering in coated pits.

ARH is an adaptor protein required for efficient endocytosis of low density lipoprotein (LDL) receptors (LDLRs) in selected tissues. Individuals lacking ARH (ARH-/-) have severe hypercholesterolemia due to impaired hepatic clearance of LDL. Immortalized lymphocytes, but not fibroblasts, from ARH-deficient subjects fail to internalize LDL. To further define the role of ARH in LDLR function, we compared the subcellular distribution of the LDLR in lymphocytes from normal and ARH-/- subjects. In normal lymphocytes LDLRs were predominantly located in intracellular compartments, whereas in ARH-/- cells the receptors were almost exclusively on the plasma membrane. Biochemical assays and quantification of LDLR by electron microscopy indicated that ARH-/- lymphocytes had >20-fold more LDLR on the cell surface and a approximately 27-fold excess of LDLR outside of coated pits. The accumulation of LDLR on the cell surface was not due to failure of receptors to localize in coated pits since the number of LDLRs in coated pits was similar in ARH-/- and normal cells. Despite the dramatic increase in cell surface receptors, LDL binding was only 2-fold higher in the ARH-/- lymphocytes. These findings indicate that ARH is required not only for internalization of the LDL.LDLR complex but also for efficient binding of LDL to the receptor and suggest that ARH stabilizes the associations of the receptor with LDL and with the invaginating portion of the budding pit, thereby increasing the efficiency of LDL internalization.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Integrins regulate Rac targeting by internalization of membrane domains.

Translocation of the small GTP-binding protein Rac1 to the cell plasma membrane is essential for activating downstream effectors and requires integrin-mediated adhesion of cells to extracellular matrix. We report that active Rac1 binds preferentially to low-density, cholesterol-rich membranes, and specificity is determined at least in part by membrane lipids. Cell detachment triggered internalization of plasma membrane cholesterol and lipid raft markers. Preventing internalization maintained Rac1 membrane targeting and effector activation in nonadherent cells. Regulation of lipid rafts by integrin signals may regulate the location of membrane domains such as lipid rafts and thereby control domain-specific signaling events in anchorage-dependent cells.

Animals↗
PubMed2003

Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic.

The principal lipids in animal cell lipid droplets are cholesterol, cholesterol ester, and triglyceride, but the protein composition of this compartment is largely unknown. Here we report on the proteomic analysis of lipid droplets. Using a combination of mass spectrometry and immunoblotting, we identify nearly 40 specifically associated proteins in droplets isolated from Chinese hamster ovary K2 cells grown in normal medium. The proteins fall in to five groups: structural molecules of the droplet-like adipose differentiation-related protein; multiple enzymes involved in the synthesis, storage, utilization, and degradation of cholesterol esters and triglycerides; multiple, different Rab GTPases known to be involved in regulating membrane traffic; signaling molecules such as p50RhoGAP; and a group of proteins that do not fit any classification but include proteins often found in caveolae/rafts such as caveolin-1 and 2 and flotillin-1. The proteome of droplets isolated from cells grown in the presence of oleate is largely the same except for an increase in the amount of adipose differentiation-related protein, caveolin-1, and a protein thought to be involved in phospholipid recycling called CGI-58. Based on the protein profile, the lipid droplet appears to be a complex, metabolically active organelle that is directly involved in membrane traffic and possibly phospholipid recycling. We propose the name adiposome for this organelle.

Animals↗
PubMed2003

A cholesterol-regulated PP2A/HePTP complex with dual specificity ERK1/2 phosphatase activity.

The acute depletion of membrane cholesterol causes the concentration of pERK1/2 in caveola/raft lipid domains and the cytosol of human fibroblasts to dramatically increase. This increase could be caused by either the activation of MEK-1 or the inhibition of a pERK phosphatase. Here we describe the isolation of a high molecular weight ( approximately 440 kDa), cholesterol-regulated pERK phosphatase that dephosphorylates both the phosphotyrosine and the phosphothreonine residues in the activation loop of the enzyme. The dual activity in the complex appears to be due to the combined activities of the serine/threonine phosphatase PP2A and the tyrosine phosphatase HePTP. Acute depletion of cholesterol causes the disassembly of the complex and a concomitant loss of the dual specificity pERK phosphatase activity. The existence of a cholesterol-regulated HePTP/PP2A activity provides a molecular explanation for why ERK activity is sensitive to membrane cholesterol levels, and raises the possibility that ERK plays a role in regulating the traffic of cholesterol to caveolae/rafts and other membranes.

Biological Transport, Active↗
PubMed2003

LRP: role in vascular wall integrity and protection from atherosclerosis.

Vascular smooth muscle cell (SMC) proliferation and migration are important events in the development of atherosclerosis. The low-density lipoprotein receptor-related protein (LRP1) mediates suppression of SMC migration induced by platelet-derived growth factor (PDGF). Here we show that LRP1 forms a complex with the PDGF receptor (PDGFR). Inactivation of LRP1 in vascular SMCs of mice causes PDGFR overexpression and abnormal activation of PDGFR signaling, resulting in disruption of the elastic layer, SMC proliferation, aneurysm formation, and marked susceptibility to cholesterol-induced atherosclerosis. The development of these abnormalities was reduced by treatment with Gleevec, an inhibitor of PDGF signaling. Thus, LRP1 has a pivotal role in protecting vascular wall integrity and preventing atherosclerosis by controlling PDGFR activation.

Animals↗
PubMed2003

Joe Goldstein and Mike Brown: from cholesterol homeostasis to new paradigms in membrane biology.

Joe Goldstein and Mike Brown have worked for over 30 years on the molecular basis of cholesterol homeostasis. Through the systematic use of genetics, biochemistry, molecular biology and cell biology, they have identified a complex set of interacting molecules that work coordinately to regulate cholesterol import and synthesis. Not only did they identify the crucial proteins in this pathway but also determined their function. An unexpected outcome of their work has been a new understanding of the structure and function of cell membranes. From the low-density lipoprotein receptor to sterol regulatory element binding protein (SREBP) to SREBP cleavage-activating protein to Insig-1, each protein has provided a new and fundamentally novel insight into how membranes function as molecular sensors that respond to changes in the metabolic condition of the cell by moving molecules between cellular compartments.*

Animals↗
PubMed2003

Function of caveolae in Ca2+ entry and Ca2+-dependent signal transduction.

The correct spatial and temporal control of Ca2+ signaling is essential for such cellular activities as fertilization, secretion, motility, and cell division. There has been a long-standing interest in the role of caveolae in regulating intracellular Ca2+ concentration. In this review we provide an updated view of how caveolae may regulate both Ca2+ entry into cells and Ca2+-dependent signal transduction

Animals↗
PubMed2003

Caveolin regulation of endothelial function.

Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling molecules critical for cell function. Recent studies have begun to identify the functions of caveolin-1, the 22-kDa caveolar protein that oligomerizes and inserts into the cytoplasmic face of the plasma membrane. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a "master regulator" of signaling molecules in caveolae. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung fibrosis, indicating its importance in cardiac and lung development. In the endothelium, caveolin-1 regulates nitric oxide signaling by binding to and inhibiting endothelial nitric oxide synthase (eNOS). Increased cytosolic Ca2+ or activation of the kinase Akt leads to eNOS activation and its dissociation from caveolin-1. Caveolae have also been proposed as the vesicle carriers responsible for transcellular transport (transcytosis) in endothelial cells. Transcytosis, the primary means of albumin transport across continuous endothelia, occurs by fission of caveolae from the membrane. This event is regulated by tyrosine phosphorylation of caveolin-1 and dynamin. As Ca2+ influx channels and pumps are localized in caveolae, caveolin-1 is also an important determinant of Ca2+ signaling in endothelial cells. Many of these findings were presented in San Diego, CA, at the 2003 Experimental Biology symposium "Caveolin Regulation of Endothelial Function" and are reviewed in this summary.

Animals↗