Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

Human cytomegalovirus (HCMV) infection of smooth muscle cells (SMCs) in vivo has been linked to a viral etiology of vascular disease. In this report, we demonstrate that HCMV infection of primary arterial SMCs results in significant cellular migration. Ablation of the chemokine receptor, US28, abrogates SMC migration, which is rescued only by expression of the viral homolog and not a cellular G protein-coupled receptor (GPCR). Expression of US28 in the presence of CC chemokines including RANTES or MCP-1 was sufficient to promote SMC migration by both chemokinesis and chemotaxis, which was inhibited by protein tyrosine kinase inhibitors. US28-mediated SMC migration provides a molecular basis for the correlative evidence that links HCMV to the acceleration of vascular disease.

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Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs) are removed from the postsynaptic plasma membrane of cultured hippocampal neurons by rapid, ligand-induced endocytosis. Although endocytosis of AMPARs can be induced by high concentrations of AMPA without concomitant activation of N-methyl-Daspartate (NMDA) receptors (NMDARs), NMDAR activation is required for detectable endocytosis induced by synaptically released glutamate. Activated AMPARs colocalize with AP2, a marker of endocytic coated pits, and endocytosis of AMPARs is blocked by biochemical inhibition of clathrin-coated pit function or overexpression of a dominant-negative mutant form of dynamin. These results establish that ionotropic receptors are regulated by dynamin-dependent endocytosis and suggest an important role of endocytic membrane trafficking in the postsynaptic modulation of neurotransmission.F ast excitatory synaptic transmission in the mammalian central nervous system is mediated primarily by ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type and Nmethyl-D-aspartate (NMDA)-type ionotropic glutamate receptors, which are coexpressed at many synapses and subserve distinct physiological functions in synaptic transmission (1-3). Although the vast majority of excitatory synapses in the hippocampus expresses functional NMDA receptors (NMDARs), electrophysiological and anatomical data suggest that the number of AMPA receptors (AMPARs) expressed at individual synapses on CA1 pyramidal cells is highly variable (4-8). Furthermore, recent evidence suggests that the surface expression of AMPARs at individual synapses is not fixed but is dynamically regulated by neuronal activity (2, 9-12). This activity-dependent regulation of the synaptic expression of AMPARs may contribute to the changes in synaptic strength that occur during NMDAR-dependent long-term potentiation and long-term depression (11, 12). Surprisingly, little is known about the detailed molecular mechanisms that regulate the number of AMPARs at excitatory synapses. Previously, we showed a pronounced redistribution of AMPARs away from synaptic sites within minutes after the triggering of long-term depression (12) or pharmacological activation of AMPARs (13). Herein, we show that this process is mediated by dynamin-dependent endocytosis and identify a role of NMDAR activation in promoting AMPAR endocytosis under physiological conditions. Materials and MethodsCell Culture and Immunocytochemistry. Hippocampal cultures were prepared as described (12, 13) and were used for experimentation at 2-3 weeks after plating. Surface AMPARs were stained with an antibody recognizing an extracellular epitope (amino acids 271-285) of the rat GluR1 subunit (Oncogene Research). Before treatment, antibody (5 g͞ml) was applied to live cells for 15 min at 37°C in conditioned cell medium. Cells were then briefly...

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