Regulation of Endocytic Clathrin Dynamics by Cargo Ubiquitination

Some endocytic cargoes control clathrin-coated pit (CCP) maturation, but it is not known how such regulation is communicated. We found that μ-opioid neuropeptide receptors signal to their enclosing CCPs by ubiquitination. Nonubiquitinated receptors delay CCPs at an intermediate stage of maturation, after clathrin lattice assembly is complete but before membrane scission. Receptor ubiquitination relieves this inhibition, effectively triggering CCP scission and producing a receptor-containing endocytic vesicle. The ubiquitin modification that conveys this endocytosis-promoting signal is added to the receptor’s first cytoplasmic loop, catalyzed by the Smurf2 ubiquitin ligase, and coordinated with activation-dependent receptor phosphorylation and clustering through Smurf2 recruitment by the endocytic adaptor beta-arrestin. Epsin1 detects the signal at the CCP and is required for ubiquitin-promoted scission. This cargo-to-coat communication system mediates a biochemical checkpoint that ensures appropriate receptor ubiquitination for later trafficking, and it controls specific receptor loading into CCPs by sensing when a sufficient quorum is reached

Internalization Dissociates β2-Adrenergic Receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β2-adrenergic receptors (β2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between β2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β2ARs and endosome markers increases. Energy transfer between β2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β2ARs associate transiently with each other in the plasma membrane, or that β2AR dimers or oligomers are actively disrupted during internalization

Subcellular localization of MC4R with ADCY3 at neuronal primary cilia underlies a common pathway for genetic predisposition to obesity.

Most monogenic cases of obesity in humans have been linked to mutations in genes encoding members of the leptin-melanocortin pathway. Specifically, mutations in MC4R, the melanocortin-4 receptor gene, account for 3-5% of all severe obesity cases in humans1-3. Recently, ADCY3 (adenylyl cyclase 3) gene mutations have been implicated in obesity4,5. ADCY3 localizes to the primary cilia of neurons 6 , organelles that function as hubs for select signaling pathways. Mutations that disrupt the functions of primary cilia cause ciliopathies, rare recessive pleiotropic diseases in which obesity is a cardinal manifestation 7 . We demonstrate that MC4R colocalizes with ADCY3 at the primary cilia of a subset of hypothalamic neurons, that obesity-associated MC4R mutations impair ciliary localization and that inhibition of adenylyl cyclase signaling at the primary cilia of these neurons increases body weight. These data suggest that impaired signaling from the primary cilia of MC4R neurons is a common pathway underlying genetic causes of obesity in humans

Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors

Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative method using a sphingosine-1-phosphate (S1P) receptor as a model. Because of a lack of suitable binding studies, it has been difficult to study S1P receptor internalisation. Using a N-terminal HisG-tag, S1P1 receptors on the cell membrane can be visualised via immunocytochemistry with a specific anti-HisG antibody. S1P-induced internalisation was concentration dependent and was quantified using a microplate reader, detecting either absorbance, a fluorescent or luminescent signal, depending on the antibodies used. Among those, the fluorescence detection method was the most convenient to use. The relative ease of this method makes it suitable to measure a large number of data points, e.g. to compare the potency and efficacy of receptor ligands

Momentum losses by charge exchange with neutral particles in H-mode discharges at JET

Introduction Extensive investigations both in theory and experiments have been done in recent years to identify the influence of rotation on plasma performance. Results have shown that a radial velocity gradient can play a role in the suppression of turbulent transport and profile stiffness [1]. It remains however largely unknown what processes determine the shape and magnitude of the observed rotation profile. With the presence of an inwards momentum pinch [2], the edge momentum density is observed to contribute significantly to the global confinement [3]. Therefore, in order to accurately predict the observed rotation profile, a better understanding of the processes that determine the edge rotation is needed. Several components play a role in momentum transport in the edge. The dominant source of torque at JET is provided by NBI, while radial transport by outwards diffusivity and an inwards convective pinch redistribute the momentum density. At the edge, a continuous sink is present in the form of charge-exchange (CX) interactions between plasma ions and a neutral particle background. Besides these direct losses, the penetration of low energy neutrals into the plasma periphery is also believed to play a role in several plasma models related to the pedestal shape [4] and the L-H transition [5]. In this paper, the results from a qualitative neutral transport model are used to assess the penetration of neutral atoms into the plasma edge. A forward model of the passive charge-exchange emission [6] is used in order to quantify the neutral density and to estimate the magnitude of momentum and energy losses by CX interactions

Differences in the signaling pathways of α1A- and α1B-adrenoceptors are related to different endosomal targeting

Aims: To compare the constitutive and agonist-dependent endosomal trafficking of α1A- and α1B-adrenoceptors (ARs) and to establish if the internalization pattern determines the signaling pathways of each subtype. Methods: Using CypHer5 technology and VSV-G epitope tagged α1A- and α1B-ARs stably and transiently expressed in HEK 293 cells, we analyzed by confocal microscopy the constitutive and agonist-induced internalization of each subtype, and the temporal relationship between agonist induced internalization and the increase in intracellular calcium (determined by FLUO-3 flouorescence), or the phosphorylation of ERK1/2 and p38 MAP kinases (determined by Western blot). Results and Conclusions: Constitutive as well as agonist-induced trafficking of α1A and α1B ARs maintain two different endosomal pools of receptors: one located close to the plasma membrane and the other deeper into the cytosol. Each subtype exhibited specific characteristics of internalization and distribution between these pools that determines their signaling pathways: α1A-ARs, when located in the plasma membrane, signal through calcium and ERK1/2 pathways but, when translocated to deeper endosomes, through a mechanism sensitive to β-arrestin and concanavalin A, continue signaling through ERK1/2 and also activate the p38 pathway. α1B-ARs signal through calcium and ERK1/2 only when located in the membrane and the signals disappear after endocytosis and by disruption of the membrane lipid rafts by methyl-β-cyclodextrin

Screening of Hydrocarbon Sources in JET

Carbon is the principal impurity in the Joint European Torus (JET). Methane screening experiments quantify the ability of the scrape-off layer (SOL)/divertor system to ionize carbon and transport it to the divertor, preventing core plasma contamination. Previous JET publications studied edge-localized-mode-averaged high-confinement mode screening, and separately, evaluated the methodology of low-confinement mode (L-mode) screening measurements. This paper extends the L-mode measurements to include relevant plasma parameter scans and DIVIMP modeling of the L-mode screening