Cavin-3 is a tumor suppressor protein of unknown function. Using both in vivo and in vitro approaches, we show that cavin-3 dictates the balance between ERK and Akt signaling. Loss of cavin-3 increases Akt signaling at the expense of ERK, while gain of cavin-3 increases ERK signaling at the expense Akt. Cavin-3 facilitates signal transduction to ERK by anchoring caveolae to the membrane skeleton of the plasma membrane via myosin-1c. Caveolae are lipid raft specializations that contain an ERK activation module and loss of the cavin-3 linkage reduces the abundance of caveolae, thereby separating this ERK activation module from signaling receptors. Loss of cavin-3 promotes Akt signaling through suppression of EGR1 and PTEN. The in vitro consequences of the loss of cavin-3 include induction of Warburg metabolism (aerobic glycolysis), accelerated cell proliferation, and resistance to apoptosis. The in vivo consequences of cavin-3 knockout are increased lactate production and cachexia.DOI: http://dx.doi.org/10.7554/eLife.00905.001

This article is available online at http://www.jlr.org by internalization of LDLR-lipoprotein complexes through clathrin-coated pits into endocytic vesicles. Nascent endocytic vesicles recruit the early endosome antigen 1 (EEA1) protein ( 1, 2 ), which is part of the molecular machinery that drives fusion of endocytic vesicles with each other and with early endosomes. Endocytic vesicles and endosomes acidify via the V-pump and lose calcium via the TRPV2 calcium channel ( 3-6 ). Both acidic pH and loss of calcium accelerate lipoprotein dissociation from the LDLR ( 7,8 ). After release, the LDLR recycles back to the surface for additional rounds of uptake, while released lipoproteins progress through the endosomal system, eventually reaching lysosomes, where hydrolysis occurs.The molecular details of how acidic pH and loss of calcium drive release are generally well understood; however, the importance of each process for release during endocytosis is not clear. The LDLR consists of seven LDLR type A (LA) repeats; two EGF-like repeats (EGF-A and EGF-B); six YWTD repeats that form a ␤ -propeller; a third EGF-like repeat (EGF-C); a single transmembrane domain; and a short cytoplasmic tail. LDL binds to the LDLR through interaction of apolipoprotein B100 (apoB100) of LDL with LA3-7 and the EGF-A modules of the LDLR ( 9, 10 ). VLDL remnants bind to the LDLR through interaction of apolipoprotein E (apoE) of remnants with LA4 and LA5 of the LDLR ( 10, 11 ). LA repeats are calcium-binding modules, and loss of calcium results in a conformational change that accelerates release ( 8,(12)(13)(14). LA4, which is required for both apoB100 and apoE binding, has particularly weak calcium-binding affi nity ( 15 ), suggesting that loss of calcium from LA4 initiates lipoprotein dissociation by the calcium release process. Acidic pH promotes loss of calcium by protonating the glutamates and aspartates that coordinate calcium in LA repeats. Acidic pH also accelerates lipoprotein release through a calcium-independent Abstract The LDL receptor (LDLR) supports effi cient uptake of both LDL and VLDL remnants by binding lipoprotein at the cell surface, internalizing lipoprotein through coated pits, and releasing lipoprotein in endocytic compartments before returning to the surface for further rounds of uptake. While many aspects of lipoprotein binding and receptor entry are well understood, it is less clear where, when, and how the LDLR releases lipoprotein. To address these questions, the current study employed quantitative fl uorescence imaging to visualize the uptake and endosomal processing of LDL and the VLDL remnant ␤ -VLDL. We fi nd that lipoprotein release is rapid, with most release occurring prior to entry of lipoprotein into early endosomes. Published biochemical studies have identifi ed two mechanisms of lipoprotein release: one that involves the ␤ -propeller module of the LDLR and a second that is independent of this module. Quantitative imaging comparing uptake supported by the normal LDLR or by an LDLR variant incapable of ␤...

Fluorescence microscopy can be used to assess quantitatively the interaction between a ligand and its receptor, between two macromolecules, or between a macromolecule and a particular intracellular compartment by co-localization analysis. In general, this analysis involves tagging potential interacting partners with distinct fluorophores-by direct labeling of a small ligand, by expression of fluorescent cDNA constructs, by immunofluorescence labeling, or by some combination of these methods. Pairwise comparison of the fluorescence intensity of the two fluorophores at each pixel in a two channel digital image of the sample reveals regions where both are present. With appropriate protocols, the image data can be interpreted to indicate where the potential interacting partners are co-localized. Keeping in mind the limited resolution of the light microscope, co-localization is often used to support the claim that two molecules are interacting.All quantitative methods for evaluating co-localization begin with identifying the pixels where the intensities of both color channels are above background. Typically this involves two sequential image segmentation steps: the first to exclude pixels where neither channel is above background, and the second to set overlap thresholds that exclude pixels where only one color channel is present. Following segmentation, various quantitative measures can be computed to describe the remaining subset of pixels where the two color channels overlap. These metrics range from simple calculation of the fraction of pixels where overlap occurs to more sophisticated image correlation metrics. Additional constraints may be employed to distinguish true co-localization from random overlap. Finally, an image map showing only the co-localized pixels may be displayed as an additional image channel in order to visualize the spatial distribution of co-localized pixels. Several commercial and open source software solutions provide this type of co-localization analysis, making image segmentation and calculation of metrics relatively straightforward. As an example, we provide a protocol for the time-dependent co-localization of fluorescently tagged lipoproteins with LDL receptor (LDLR) and with the early endosome marker EEA1.

This article is available online at http://www.jlr.org (ARH) and the disabled-like protein 2 (dab2). Both ARH and dab2 target LDLRs to coated pits through binding sites for the LDLR, clathrin, and the adaptor protein 2 (AP-2) complex. ARH binds to the FDNPVY 807 sequence of the LDLR through a phosphotyrosine-binding (PTB) domain, to the heavy chain of clathrin through a clathrin box sequence, and to the ␤ 2-subunit of AP-2 through a sequence with strong homology to the AP-2-binding sequences of ␤ -arrestins ( 5-8 ) ( Fig. 1A ). Y807 of the FDNPVY 807 plays a critical role in the interaction of ARH and dab2 with the LDLR, and mutation of Y807 to either cysteine or alanine cripples LDLR-dependent LDL uptake ( 3,5,8,9 ). By contrast, uptake of VLDL remnants does not require ARH, dab2, or a functional FDNPVY sequence because binding of VLDL remnants to the LDLR induces a separate endocytic process that involves a second internalization motif on the LDLR cytoplasmic domain ( 10 ). It is not clear why the LDLR needs an induced process for VLDL remnant uptake when the FDNPVY-dependent process can internalize both LDL and VLDL remnants, nor why the FDNPVY process utilizes both the ARH and dab2 adaptors when either adaptor is suffi cient to support lipoprotein uptake by the LDLR.A key question is why an ARH is necessary. ARH has highest expression in kidney, liver, and placenta ( 11 ); however, dab2 is also highly expressed in both kidney and placenta, and it is dab2, not ARH, that is required for normal placental and kidney function ( 12 ). Hepatocytes and peripheral blood leukocytes lack dab2 ( 13,14 ), and ARH defi ciency in both humans and mice sharply reduces LDL clearance rates, resulting in hypercholesterolemia ( 15-18 ). Why hepatocytes and leukocytes normally rely upon ARH is unclear because both cell types lose their dependence on ARH for LDL uptake when dab2 expression is induced Abstract The LDL receptor (LDLR) relies upon endocytic adaptor proteins for internalization of lipoproteins. The results of this study show that the LDLR adaptor autosomal recessive hypercholesterolemia protein (ARH) requires nitric oxide to support LDL uptake. Nitric oxide nitrosylates ARH at C199 and C286, and these posttranslational modifi cations are necessary for association of ARH with the adaptor protein 2 (AP-2) component of clathrin-coated pits. In the absence of nitrosylation, ARH is unable to target LDL-LDLR complexes to coated pits, resulting in poor LDL uptake. The role of nitric oxide on LDLR function is specifi c for ARH because inhibition of nitric oxide synthase activity impairs ARH-supported LDL uptake but has no effect on other LDLR-dependent lipoprotein uptake processes, including VLDL remnant uptake and dab2-supported LDL uptake. These fi ndings suggest that cells that depend upon ARH for LDL uptake can control which lipoproteins are internalized by their LDLRs through changes in nitric oxide. -Zhao, Z., S. Pompey, H. Dong, J. Weng, R. Garuti, and P. Michaely. The LDL receptor (LDLR) internalizes a broad spectrum ...