In the nucleus accumbens (NAc), a key structure to the effects of all addictive drugs, presynaptic cannabinoid CB1 receptors (CB1Rs) and postsynaptic metabotropic glutamate 5 receptors (mGluR5s) are the principal effectors of endocannabinoid (eCB)-mediated retrograde long-term depression (LTD) (eCB-LTD) at the prefrontal cortex-NAc synapses. Both CB1R and mGluR5 are involved in cocaine-related behaviors; however, the impact of in vivo cocaine exposure on eCB-mediated retrograde synaptic plasticity remains unknown. Electrophysiological and biochemical approaches were used, and we report that a single in vivo cocaine administration abolishes eCB-LTD. This

Abstract. Members of the cytokine receptor family are composed of several noncovalently linked chains with sequence and structure homologies in their extracellular domain. Receptor subfamily members share at least one component: thus the receptors for interleukin (IL) 2 and ILl5 have common ~ and 3" chains, while those for IL2, 4, 7, and 9 have a common 3' chain. The intracellular pathway followed by IL2 receptors after ligand binding and endocytosis was analyzed by immunofluorescence and confocal microscopy in a human T lymphocytic cell line. Surprisingly, the a, ~, and 3' chains had different intracellular localizations after being endocytosed together. The ct chain was always in transferl-in-positive compartments (early/recycling endosomes), both at early and late internalization times, but was never detected in rab7-positive compartments (late endosomes). On the other hand, at late internalization times, the/3 and 3, chains were excluded from transferrin-positive organelles and did not colocalize with ~. Furthermore, /3 could be found in rab7-positive vesicles. These differences suggest that the ot chain recycles to the plasma membrane, while the ~ and 3' chains are sorted towards the degradation pathway. The half-lives of these three chains on the cell surface also reflect their different intracellular fates after endocytosis. The and 3' chains are very short-lived polypeptides since their half-life on the surface is only =1 h, whereas c~ is a much more stable surface protein. This shows for the first time that components of a multimeric receptor can be sorted separately along the endocytic pathway.

The dynamins comprise a large family of mechanoenzymes known to participate in membrane modeling events. All three conventional dynamin genes (Dyn1, Dyn2, Dyn3) are expressed in mammalian brain and produce more than 27 different dynamin proteins as a result of alternative splicing. Past studies have suggested that Dyn1 participates in specialized neuronal functions such as rapid synaptic vesicle recycling, while Dyn2 may mediate the conventional clathrin-mediated uptake of surface receptors. Currently, the distribution, expression, and function of Dyn3 in neurons, or in any other cell type, are completely undefined. Here, we demonstrate that Dyn1 and Dyn3 localize differentially in the synapse. Dyn1 concentrates within the presynaptic compartment, while Dyn3 localizes to dendritic spine tips. Within the postsynaptic density (PSD), we found Dyn3, but not Dyn1, to be part of a biochemically isolated complex comprised of Homer and metabotropic glutamate receptors. Finally, although dominant-negative Dyn3 did not seem to inhibit receptor endocytosis, overexpression of a specific Dyn3 spliced variant in mature neurons caused a marked remodeling of dendritic spines. These data suggest that Dyn3 is a postsynaptic dynamin and, like its binding partner Homer, plays a significant role in dendritic spine morphogenesis and remodeling.

Glutamate receptors are clustered at the membrane through interactions with intracellular scaffolding proteins and cytoskeletal elements but can also be found in intracellular compartments or dispersed in the membrane. This distribution results from an equilibrium between the different pools of receptors whose dynamic is poorly known. The group I metabotropic glutamate receptor 5 (mGluR5) is concentrated in an annulus around the postsynaptic density but also found in large amounts in the extrasynaptic membrane. To analyze the dynamic of stabilization of mGluR5, we used single-particle tracking, force measurements, and fluorescence recovery to measure the mobility of mGluR5. We found that receptor activation increases receptor diffusion, whereas the scaffolding protein Homer favors confinement of receptor movements within clusters of Homer-mGluR5. However, this stabilization is reversible, because even in the presence of Homer, receptors still enter and exit from clusters at fast rates. Furthermore, clusters themselves are highly dynamic both in their movements and in their composition, which can vary within tens of seconds. Thus, exchange of receptors between dispersed and clustered states is fast and regulated during physiological processes. These properties may explain certain fast changes in receptor composition observed at postsynaptic densities.

No abstract

The c-ets-1 proto-oncogene and the related c-ets-2 gene encode related nuclear chromatin-associated proteins which bind DNA in vitro. To investigate the possibility that Etsl and Ets2 are transcriptional activators, we analyzed the ability of these proteins to trans-activate promoter/enhancer sequences in transient co-transfection experiments. A CAT construct driven by the long terminal repeat of the human T cell leukemia virus, HTLV-1 was found to be trans-activated by both Etsl and Ets2 in NIH3T3 and HeLa cells. The increased levels of CAT activity were paralleled by increased levels of correctly initiated CAT mRNA. Mutant Etsl proteins unable to accumulate in the nucleus were found to be inactive. An ets-responsive sequence between positions -117 and -160 of the LTR was identified by analyses of a series of 5' deletion mutants of the HTLV-1 LTR and of dimerized versions of specific motifs of the LTR enhancer region. Using a gel shift binding assay, Etsl was found to bind specifically to an oligonucleotide corresponding to region -117 to -160. This sequence, which also contributes to Taxl responsiveness of the HTLV-1 LTR, is characterized by the presence of four repeats of a pentanucleotide sequence of the type CC(T/A)CC. Competition

Metabotropic glutamate receptors 5 (mGluR5) are members of the growing group C G protein-coupled receptor family. Widely expressed in mammalian brain, they are involved in modulation of the glutamate transmission. By means of transfection of mGluR5 receptors in COS-7 cells and primary hippocampal neurons in culture followed by immunocytochemistry and quantitative image analysis and by a biochemical assay, we have studied the internalization of mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well as receptors in which endogenous activity had been inhibited by an inverse agonist. We have measured a constitutive rate of endocytosis of 11.7%/min for mGluR5a. We report for the first time the endocytosis pathway of mGluR5. Internalization of mGluR5 is not mediated by clathrin-coated pits. Indeed, inhibition of this pathway by Eps15 dominant negative mutants did not disturb their endocytosis. However, the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5 in COS-7. mGluR5 is the first shown member of the group C G-protein coupled receptor family internalized by a nonconventional pathway.Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. It exerts its effects by binding to ionotropic and metabotropic receptors. Metabotropic glutamate receptors (mGluRs) 1 belong to the seven-transmembrane domain G protein-coupled receptor (GPCR) family. Together with calcium-sensing receptors, ␥-aminobutyric acid receptor B (GABA B ) and pheromone, olfactory and taste receptors, mGluRs form the third family of GPCRs (group C) (for reviews, see Refs. 1 and 2). They have little homology with other GPCRs from groups A and B and are characterized by a large N-terminal extracellular domain that binds glutamate. Eight genes code for mGluRs that generate more than 15 proteins by alternative splicing. On the basis of their sequence similarity, pharmacology, and signal transduction mechanisms, mGluRs have been classified into three groups. Group 1 consists of mGluR1 and mGluR5 that are preferentially positively coupled to phospholipase C via G q/11 proteins. mGluRs of group 2 and 3 are negatively coupled to adenylate cyclase. Group 1 mGluRs are expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in several forms of synaptic plasticity.Currently, very little is known about the targeting and trafficking mechanisms of mGluRs in cells. During the last few years, attention has focused mainly on endocytosis of a subtype of ionotropic glutamate receptors: the ␣-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptors. Internalization of AMPA receptors in response to various stimuli is an important process for the rapid attenuation of neurotransmission during synaptic plasticity (3). By different approaches, it has been s...

No abstract