Biomedical subjects
Publications and source records attributed to M von Zastrow.
Understanding how biochemical pathways are connected in the cell is one of the big challenges facing cell biologists. In a Perspective, von Zastrow and Mostov describe new work that identifies a protein called RGS-PX1 as the linchpin that connects signal transduction activated by G protein-coupled receptors with membrane trafficking events.
The beta(2)-adrenergic receptor and delta opioid receptor represent distinct G protein-coupled receptors that undergo agonist-induced endocytosis via clathrin-coated pits but differ significantly in their postendocytic sorting between recycling and degradative membrane pathways, respectively. Previous results indicate that a distal portion of the carboxyl-terminal cytoplasmic domain of the beta(2)-adrenergic receptor, which engages in PDZ domain-mediated protein interaction, is required for efficient recycling of receptors after agonist-induced endocytosis. Here we demonstrate that a four-residue sequence (DSLL) comprising the core of this protein interaction domain functions as a transplantable endocytic sorting signal that is sufficient to re-route endocytosed delta opioid receptor into a rapid recycling pathway, to inhibit proteolytic down-regulation of receptors, and to mediate receptor-autonomous sorting of mutant receptors from the wild type allele when co-expressed in the same cells. These observations define a transplantable signal mediating rapid recycling of a heterologous G protein-coupled receptor, and they suggest that rapid recycling of certain membrane proteins does not occur by bulk membrane flow but is instead mediated by a specific endocytic sorting mechanism.
The delta-opioid receptor (DOR) can undergo proteolytic down-regulation by endocytosis of receptors followed by sorting of internalized receptors to lysosomes. Although phosphorylation of the receptor is thought to play an important role in controlling receptor down-regulation, previous studies disagree on whether phosphorylation is actually required for the agonist-induced endocytosis of opioid receptors. Furthermore, no previous studies have determined whether phosphorylation is required for subsequent sorting of internalized receptors to lysosomes. We have addressed these questions by examining the endocytic trafficking of a series of mutant versions of DOR expressed in stably transfected HEK 293 cells. Our results confirm that phosphorylation is not required for agonist-induced endocytosis of truncated mutant receptors that lack the distal carboxyl-terminal cytoplasmic domain containing sites of regulatory phosphorylation. However, phosphorylation is required for endocytosis of full-length receptors. Mutation of all serine/threonine residues located in the distal carboxyl-terminal tail domain of the full-length receptor to alanine creates functional mutant receptors that exhibit no detectable agonist-induced endocytosis. Substitution of these residues with aspartate restores the ability of mutant receptors to undergo agonist-induced endocytosis. Studies using green fluorescent protein-tagged versions of arrestin-3 suggest that the distal tail domain, when not phosphorylated, inhibits receptor-mediated recruitment of beta-arrestins to the plasma membrane. Biochemical and radioligand binding studies indicate that, after endocytosis occurs, phosphorylation-defective mutant receptors traffic to lysosomes with similar kinetics as wild type receptors. We conclude that phosphorylation controls endocytic trafficking of opioid receptors primarily by regulating a "brake" mechanism that prevents endocytosis of full-length receptors in the absence of phosphorylation. After endocytosis occurs, subsequent steps of membrane trafficking mediating sorting and transport to lysosomes do not require receptor phosphorylation.
We have observed an unexpected type of nonreciprocal "cross-regulation" of the agonist-induced endocytosis of G protein-coupled receptors by clathrin-coated pits. Isoproterenol-dependent internalization of beta2-adrenergic receptors in stably transfected HEK293 cells was specifically blocked (>65% inhibition) by vasopressin-induced activation of V2 vasopressin receptors co-expressed at similar levels. In contrast, activation of beta2 receptors caused no detectable effect on V2 receptor internalization in the same cells. Several pieces of evidence suggest that this nonreciprocal inhibition of endocytosis is mediated by receptor-specific intracellular trafficking of beta-arrestins. First, previous studies showed that the activation of V2 but not beta2 receptors caused pronounced recruitment of beta-arrestins to endocytic membranes (Oakley, R. H., Laporte, S. A., Holt, J. A., Barak, L. S., and Caron, M. G. (1999) J. Biol. Chem. 274, 32248-32257). Second, overexpression of arrestin 2 or 3 (beta-arrestin 1 or 2) abolished the V2 receptor-mediated inhibition of beta2 receptor internalization. Third, mutations of the V2 receptor that block endomembrane recruitment of beta-arrestins eliminated the V2 receptor-dependent blockade of beta2 receptor internalization. These results identify a novel type of heterologous regulation of G protein-coupled receptors, define a new functional role of receptor-specific intracellular trafficking of beta-arrestins, and suggest an experimental method to rapidly modulate the functional activity of beta-arrestins in intact cells.
Many G-protein-coupled receptors (GPCRs) are regulated by endocytosis. Pharmacological studies defining distinct processes of ligand-induced sequestration and down-regulation provided early evidence for significant complexity in the endocytic membrane trafficking of GPCRs. In this review, we discuss our present understanding of the diversity and specificity of GPCR endocytic membrane trafficking, focusing primarily on proteolytic down-regulation of GPCRs via delivery to lysosomes. We discuss evidence suggesting that certain GPCRs can be targeted selectively to lysosomes after endocytosis by the same membrane pathway that mediates the process of rapid sequestration, and we highlight recent progress in understanding a mechanism that controls the sorting of a specific GPCR between distinct membrane pathways after endocytosis by clathrin-coated pits.
Many G-protein-coupled receptors (GPCRs) undergo agonist-induced endocytosis. Such endocytosis has been implicated in diverse processes of receptor regulation, including reversible sequestration of receptors in endosomes and proteolytic downregulation of receptors in lysosomes. The precise relationships between membrane pathways that mediate receptor sequestration and downregulation remain controversial. Recent studies suggest that GPCRs can be segregated within distinct microdomains of the plasma membrane before endocytosis occurs, and others suggest that certain GPCRs are sorted between divergent membrane pathways after endocytosis by clathrin-coated pits. Furthermore, emerging data implicate a specific role of the actin cytoskeleton and receptor phosphorylation in controlling endocytic sorting of a particular GPCR. In this article, recent research into endocytosis of GPCRs will be discussed together with some important and unresolved questions regarding the diversity and specificity of mechanisms that mediate downregulation of GPCRs.
The number of G protein-coupled receptors (GPCRs) displayed at the cell surface is a critical determinant of physiological responsiveness to native ligands and drugs. Downregulation of the number of adrenergic and dopaminergic receptors present on specific neurons can be induced by receptor agonists or by drugs that increase extracellular concentrations of catecholamines such as dopamine. Thus agonist-induced downregulation of GPCRs is of potentially great importance to the treatment of Parkinson's Disease. However, little is known about biochemical mechanisms that mediate GPCR downregulation. Recent studies of cloned GPCRs have provided exciting insights into specific mechanisms that control endocytosis of receptors from the plasma membrane and regulate proteolytic degradation of receptors. In this review we briefly survey representative studies establishing that multiple mechanisms of GPCR membrane trafficking can function in downregulation function both in neural and non-neural cell types. Then we focus on our present view of mechanisms mediating regulated proteolysis of GPCRs, highlighting recent progress in understanding membrane trafficking of GPCRs from the cell surface to lysosomes. Finally we discuss emerging evidence regarding a specific mechanism that modulates sorting of certain GPCRs between recycling and degradative pathways.
Activity-mediated changes in the strength of synaptic communication are important for the establishment of proper neuronal connections during development and for the experience-dependent modification of neural circuitry that is believed to underlie all forms of behavioural plasticity. Owing to the wide-ranging significance of synaptic plasticity, considerable efforts have been made to identify the mechanisms by which synaptic changes are triggered and expressed. New evidence indicates that one important expression mechanism of several long-lasting forms of synaptic plasticity might involve the physical transport of AMPA-type glutamate receptors in and out of the synaptic membrane. Here, we focus on the rapidly accumulating evidence that AMPA receptors undergo regulated endocytosis, which is important for long-term depression.
Many G-protein-coupled receptors (GPCRs) undergo agonist-induced endocytosis. Endocytosis contributes to distinct processes that regulate the number and functional activity of receptors present in the plasma membrane, contributing to the well described processes of receptor sequestration and down-regulation. Emerging evidence suggests additional functions of endocytosis in mediating GPCR signalling via certain effector pathways, such as mitogen-activated protein kinase modules. The diverse functions of endocytosis raise fundamental questions about the nature of the vesicular carriers and membrane pathways that mediate the endocytic trafficking of specific GPCRs. Insights into the biochemical and functional properties of endocytic vesicles containing internalized opioid and adrenergic receptors will be discussed. Progress towards understanding the mechanisms that control the specificity with which distinct GPCRs are sorted to specialized subpopulations of endocytic vesicles will be highlighted.
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In certain circumstances, internalized receptors are able to continue signaling after endocytosis. Whistler et al. discuss how the interaction of heterotrimeric GTP-binding protein (G protein)-coupled receptors with arrestins and their subsequent endocytosis may contribute to activation of the mitogen-activated protein kinase pathway. This perspective delves into the role that scaffolds may play in organizing and specifying downstream signaling events that occur after internalization of G protein-coupled receptors.
The beta(2)-adrenergic receptor (B2AR) and delta-opioid receptor (DOR) are structurally distinct G protein-coupled receptors (GPCRs) that undergo rapid, agonist-induced internalization by clathrin-coated pits. We have observed that these receptors differ substantially in their membrane trafficking after endocytosis. B2AR expressed in stably transfected HEK293 cells exhibits negligible (<10%) down-regulation after continuous incubation of cells with agonist for 3 h, as assessed both by radioligand binding (to detect functional receptors) and immunoblotting (to detect total receptor protein). In contrast, DOR exhibits substantial (>/=50%) agonist-induced down-regulation when examined by similar means. Degradation of internalized DOR is sensitive to inhibitors of lysosomal proteolysis. Flow cytometric and surface biotinylation assays indicate that differential sorting of B2AR and DOR between distinct recycling and non-recycling pathways (respectively) can be detected within approximately 10 min after endocytosis, significantly before the onset of detectable proteolytic degradation of receptors ( approximately 60 min after endocytosis). Studies using pulsatile application of agonist suggest that after this sorting event occurs, later steps of membrane transport leading to lysosomal degradation of receptors do not require the continued presence of agonist in the culture medium. These observations establish that distinct GPCRs differ significantly in endocytic membrane trafficking after internalization by the same membrane mechanism, and they suggest a mechanism by which brief application of agonist can induce substantial down-regulation of receptors.
Major advances have been made in understanding mechanisms mediating downregulation of G protein-coupled receptors. Recent studies emphasize the role of multiple proteolytic mechanisms in downregulation. A specific mechanism of downregulation, mediated by endocytosis of receptors via clathrin-coated pits followed by sorting to lysosomes, has been examined in detail. Specific protein interactions that control the specificity of G-protein-coupled receptor trafficking in this pathway are beginning to be elucidated.
The endocytosis of AMPA receptors is thought to be important in the expression of long-term depression (LTD) triggered by NMDA receptor activation. Although signaling pathways necessary for LTD induction have been identified, those responsible for the regulated internalization of AMPA receptors are unknown. Here we show that activation of NMDA receptors alone can trigger AMPA receptor endocytosis through calcium influx and activation of the calcium-dependent protein phosphatase calcineurin. A distinct signaling mechanism mediates the AMPA receptor endocytosis stimulated by insulin. These results demonstrate that although multiple signaling pathways can induce AMPA receptor internalization, NMDA receptor activation enhances AMPA receptor endocytosis via a signaling mechanism required for the induction of LTD.
Little is known about the mechanisms that regulate the number of ionotropic glutamate receptors present at excitatory synapses. Herein, we show that GluR1-containing alpha-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-D-aspartate (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.
A fundamental question in cell biology is how membrane proteins are sorted in the endocytic pathway. The sorting of internalized beta2-adrenergic receptors between recycling endosomes and lysosomes is responsible for opposite effects on signal transduction and is regulated by physiological stimuli. Here we describe a mechanism that controls this sorting operation, which is mediated by a family of conserved protein-interaction modules called PDZ domains. The phosphoprotein EBP50 (for ezrinradixin-moesin(ERM)-binding phosphoprotein-50) binds to the cytoplasmic tail of the beta2-adrenergic receptor through a PDZ domain and to the cortical actin cytoskeleton through an ERM-binding domain. Disrupting the interaction of EBP50 with either domain or depolymerization of the actin cytoskeleton itself causes missorting of endocytosed beta2-adrenergic receptors but does not affect the recycling of transferrin receptors. A serine residue at position 411 in the tail of the beta2-adrenergic receptor is a substrate for phosphorylation by GRK-5 (for G-protein-coupled-receptor kinase-5) and is required for interaction with EBP50 and for proper recycling of the receptor. Our results identify a new role for PDZ-domain-mediated protein interactions and for the actin cytoskeleton in endocytic sorting, and suggest a mechanism by which GRK-mediated phosphorylation could regulate membrane trafficking of G-protein-coupled receptors after endocytosis.
Dynamin plays a critical role in the membrane fission mechanism that mediates regulated endocytosis of many G protein-coupled receptors. In addition, dynamin is required for ligand-induced activation of mitogen-activated protein kinase by certain receptors, raising a general question about the role of dynamin in mitogenic signal transduction. Here we report that endocytosis of mu and delta opioid receptors is not required for efficient ligand-induced activation of mitogen-activated protein kinase. Nevertheless, mitogenic signaling mediated by these receptors is specifically dynamin-dependent. Thus a functional role of dynamin in mitogenic signaling can be dissociated from its role in receptor-mediated endocytosis, suggesting a previously unidentified and distinct role of dynamin in signal transduction by certain G protein-coupled receptors.
We have examined the membrane localization of an AMPA receptor subunit (GluR1) and an NMDA receptor subunit (NR1) endogenously expressed in primary cultures of rat hippocampal neurons. In unstimulated cultures, both GluR1 and NR1 subunits were concentrated in SV2-positive synaptic clusters associated with dendritic shafts and spines. Within 5 min after the addition of 100 microM glutamate to the culture medium, a rapid and selective redistribution of GluR1 subunits away from a subset of synaptic sites was observed. This redistribution of GluR1 subunits was also induced by AMPA, did not require NMDA receptor activation, did not result from ligand-induced neurotoxicity, and was reversible after the removal of agonist. The activation-induced redistribution of GluR1 subunits was associated with a pronounced (approximately 50%) decrease in the frequency of miniature EPSCs, consistent with a role of GluR1 subunit redistribution in mediating rapid regulation of synaptic efficacy. We conclude that ionotropic glutamate receptors are regulated in native neurons by rapid, subtype-specific membrane trafficking, which may modulate synaptic transmission in response to physiological or pathophysiological activation.