We directly resolved discrete exocytic fusion events mediating insertion of AMPA-type glutamate receptors (AMPARs) to the somatodendritic surface of rat hippocampal pyramidal neurons, in slice and dissociated cultures, using protein tagging with a pH-sensitive GFP (green fluorescent protein) variant and rapid (10 frames/s) fluorescence microscopy. AMPAR-containing exocytic events occurred under basal culture conditions in both the cell body and dendrites; potentiating chemical stimuli produced an NMDA receptor-dependent increase in the frequency of individual exocytic events. The number of AMPARs inserted per exocytic event, estimated using singlemolecule analysis, was quite uniform but individual events differed significantly in kinetic properties affecting the subsequent surface distribution of receptors. "Transient" events, from which AMPARs dispersed laterally immediately after surface insertion, generated a pronounced but short-lived (dissipating within ϳ1 s) increase in surface AMPAR fluorescence extending locally (2-5 m) from the site of exocytosis. "Persistent" events, from which inserted AMPARs dispersed slowly (typically over 5-10 s), affected local surface receptor concentration to a much smaller degree. Both modes of exocytic insertion occurred throughout the dendritic shaft, but remarkably, neither mode of insertion was observed directly into synaptic spines. AMPARs entered spines preferentially from transient events occurring in the adjoining dendritic shaft, driven apparently by mass action and short-range lateral diffusion, and locally delivered AMPARs remained mostly in the mobile fraction. These results suggest a highly dynamic mechanism for both constitutive and activitydependent surface delivery of AMPARs, mediated by kinetically distinct exocytic modes that differ in propensity to drive lateral entry of receptors to nearby synapses.

The identification and cloning of the two major cannabinoid (CB1 and CB2) receptors together with the discovery of their endogenous ligands in the late 80s and early 90s, resulted in a major effort aimed at understanding the mechanisms and physiological roles of the endocannabinoid system (ECS). Due to its expression and localization in the central nervous system (CNS), the CB1 receptor together with its endogenous ligands (endocannabinoids (eCB)) and the enzymes involved in their synthesis and degradation, has been implicated in multiple pathophysiological events ranging from memory deficits to neurodegenerative disorders among others. In this review, we will provide a general overview of the ECS with emphasis on the CB1 receptor in health and disease. We will describe our current understanding of the complex aspects of receptor signaling and trafficking, including the non-canonical signaling pathways such as those mediated by β-arrestins within the context of functional selectivity and ligand bias. Finally, we will highlight some of the disorders in which CB1 receptors have been implicated. Significant knowledge has been achieved over the last 30 years. However, much more research is still needed to fully understand the complex roles of the ECS, particularly in vivo and to unlock its true potential as a source of therapeutic targets.

Adenosine deaminases that act on RNA are a conserved family of enzymes that catalyze a natural process of site-directed mutagenesis. Biochemically, they convert adenosine to inosine, a nucleotide that is read as guanosine during translation; thus when editing occurs in mRNAs, codons can be recoded and the changes can alter protein function. By removing the endogenous targeting domains from human adenosine deaminase that acts on RNA 2 and replacing them with an antisense RNA oligonucleotide, we have engineered a recombinant enzyme that can be directed to edit anywhere along the RNA registry. Here we demonstrate that this enzyme can efficiently and selectively edit a single adenosine. As proof of principle in vitro, we correct a premature termination codon in mRNAs encoding the cystic fibrosis transmembrane conductance regulator anion channel. In Xenopus oocytes, we show that a genetically encoded version of our editase can correct cystic fibrosis transmembrane conductance regulator mRNA, restore fulllength protein, and reestablish functional chloride currents across the plasma membrane. Finally, in a human cell line, we show that a genetically encoded version of our editase and guide RNA can correct a nonfunctional version of enhanced green fluorescent protein, which contains a premature termination codon. This technology should spearhead powerful approaches to correcting a wide variety of genetic mutations and fine-tuning protein function through targeted nucleotide deamination.

Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo-and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright "puffs" of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (Ϸtwofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo. INTRODUCTIONMembrane trafficking pathways play a fundamental role in shaping cellular responses to receptor-mediated signals. This relationship is clear when one examines endocytic trafficking of transmembrane signaling receptors, such as G protein-coupled receptors (GPCRs), for which the cellular response depends on the number of functional receptors available at the cell surface (Lefkowitz et al., 1998;Moore et al., 2007;von Zastrow and Sorkin, 2007). Removal of surface receptors by endocytosis is one mechanism by which cells can terminate or rapidly attenuate cellular responses to an activating ligand. Subsequent trafficking of internalized receptors to lysosomes promotes receptor proteolysis, typically resulting in a prolonged attenuation of cellular signaling responsiveness (Marchese et al., 2008). Recycling of internalized receptors to the plasma membrane, in contrast, restores the complement of surface receptors and can contribute to rapid recovery of functional signaling (Carman and Benovic, 1998;Ferguson, 2001;Hanyaloglu and von Zastrow, 2008). Despite the established importance of endocytic trafficking in controlling receptor-mediated signaling, relatively little is known about whether or how signaling receptors regulate the endocytic pathway.The beta-2 adrenergic receptor (B2AR) is a seven-transmembrane G protein-coupled receptor, thus representing the largest known family o...

sees chromosomes moving long distances-and not during mitosis. Chuang, Andrew Belmont, and colleagues, now unveil the inducible long-range migration of an interphase chromosome site. An hour or two after mitosis, chromosomes appear to be locked down, as only short-range, tethered diffusion-like movements had been seen during interphase. Yet several groups had noted that certain genes localize at the nuclear periphery when repressed but in the interior when active. Chuang and colleagues visualized the movements accompanying this repositioning. They used an engineered locus that they could turn on through inducible targeting of a transcriptional activator. Upon transcription factor targeting, the group observed rapid, directed movements of the locus from the nuclear envelope toward the interior. The linear paths taken, often approximately perpendicular to the envelope, discount simple diffusion. During this motion, the locus moved at speeds approaching anaphase chromosome separation. Although the existence of a nuclear actin network is still controversial, the group found a dependence of this long-range motion on actin and myosin. If the DNA is indeed transported on actin fi laments, the preferential inward movements suggest that the intranuclear actin network is polarized. The authors are now testing whether the locus returns as directly to the periphery when the transcriptional activator is removed.

Endocytosis of Na + ,K + -ATPase molecules in response to G protein-coupled receptor stimulation requires activation of class I A phosphoinositide-3 kinase (PI3K-I A ) in a protein kinase C-dependent manner. In this paper, we report that PI3K-I A , through its p85α subunit-SH3 domain, binds to a proline-rich region in the Na + ,K + -ATPase catalytic α subunit. This interaction is enhanced by protein kinase C-dependent phosphorylation of a serine residue that flanks the proline-rich motif in the Na + ,K + -ATPase α subunit and results in increased PI3K-I A activity, an effect necessary for adaptor protein 2 binding and clathrin recruitment. Thus, Ser-phosphorylation of the Na + ,K + -ATPase catalytic subunit serves as an anchor signal for regulating the location of PI3K-I A and its activation during Na + ,K + -ATPase endocytosis in response to G protein-coupled receptor signals.

G protein-coupled receptors (GPCRs) are major transducers of external stimuli and key therapeutic targets in many pathological conditions. When activated by different ligands, one receptor can elicit multiple signaling cascades that are mediated by G proteins or β-arrestin, a process defined as functional selectivity or ligand bias. However, the dynamic mechanisms underlying β-arrestin signaling remain unknown. Here, by studying the cannabinoid 1 receptor (CB1R), we identify ligand-specific endocytic dwell times, that is, the time during which receptors are clustered into clathrin pits together with β-arrestins before endocytosis, as the mechanism controlling β-arrestin signaling. Agonists inducing short endocytic dwell times produce little or no β-arrestin signaling, whereas those eliciting prolonged dwell times induce robust signaling. Remarkably, extending CB1R dwell times by preventing endocytosis substantially increased β-arrestin signaling. These studies reveal how receptor activation translates into β-arrestin signaling and identify a mechanism to control this pathway.

Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling.