Clathrin-mediated Receptor Endocytosis Research Articles

Highly Modular Protein Micropatterning Sheds Light on the Role of Clathrin-Mediated Endocytosis for the Quantitative Analysis of Protein-Protein Interactions in Live Cells.

Protein micropatterning is a powerful tool for spatial arrangement of transmembrane and intracellular proteins in living cells. The restriction of one interaction partner (the bait, e.g., the receptor) in regular micropatterns within the plasma membrane and the monitoring of the lateral distribution of the bait’s interaction partner (the prey, e.g., the cytosolic downstream molecule) enables the in-depth examination of protein-protein interactions in a live cell context. This study reports on potential pitfalls and difficulties in data interpretation based on the enrichment of clathrin, which is a protein essential for clathrin-mediated receptor endocytosis. Using a highly modular micropatterning approach based on large-area micro-contact printing and streptavidin-biotin-mediated surface functionalization, clathrin was found to form internalization hotspots within the patterned areas, which, potentially, leads to unspecific bait/prey protein co-recruitment. We discuss the consequences of clathrin-coated pit formation on the quantitative analysis of relevant protein-protein interactions, describe controls and strategies to prevent the misinterpretation of data, and show that the use of DNA-based linker systems can lead to the improvement of the technical platform.

RNAi screen reveals synthetic lethality between cyclin G-associated kinase and FBXW7 by inducing aberrant mitoses

Background:F-box and WD40 repeat domain-containing 7 (FBXW7) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of multiple oncogenic substrates. The tumour suppressor function is frequently lost in multiple cancers through genetic deletion and mutations in a broad range of tumours. Loss of FBXW7 functionality results in the stabilisation of multiple major oncoproteins, culminating in increased cellular proliferation and pro-survival pathways, cell cycle deregulation, chromosomal instability and altered metabolism. Currently, there is no therapy to specifically target FBXW7-deficient tumours.Methods:We performed a siRNA kinome screen to identify synthetically lethal hits to FBXW7 deficiency.Results:We identified and validated cyclin G-associated kinase (GAK) as a potential new therapeutic target. Combined loss of FBXW7 and GAK caused cell cycle defects, formation of multipolar mitoses and the induction of apoptosis. The synthetic lethal mechanism appears to be independent of clathrin-mediated receptor endocytosis function of GAK.Conclusions:These data suggest a putative therapeutic strategy for a large number of different types of human cancers with FBXW7 loss, many of which have a paucity of molecular abnormalities and treatment options.

The ShcD phosphotyrosine adaptor subverts canonical EGF receptor trafficking.

Shc family signalling adaptors connect activated transmembrane receptors to proximal effectors, and most also contain a sequence involved in clathrin-mediated receptor endocytosis. Notably, this AP2 adaptin-binding motif (AD) is absent from the ShcD (also known as Shc4) homolog, which also uniquely promotes ligand-independent phosphorylation of the epidermal growth factor receptor (EGFR). We now report that cultured cells expressing ShcD exhibit reduced EGF uptake, commensurate with a decrease in EGFR surface presentation. Under basal conditions, ShcD colocalises with the EGFR and facilitates its phosphorylation, ubiquitylation and accumulation in juxtanuclear vesicles identified as Rab11-positive endocytic recycling compartments. Accordingly, ShcD also functions as a constitutive binding partner for the E3 ubiquitin ligase Cbl. EGFR phosphorylation and focal accumulation likewise occur upon ShcD co-expression in U87 glioma cells. Loss of ShcD phosphotyrosine-binding function or insertion of the ShcA AD sequence each restore ligand acquisition through distinct mechanisms. The AD region also contains a nuclear export signal, indicating its multifunctionality. Overall, ShcD appears to possess several molecular permutations that actively govern the EGFR, which may have implications in development and disease.

Acetylation as a mechanism that regulates axonal regeneration.

The capacity for adult axons to regenerate after injury is diminished compared with developing axons. In the case of central nervous system (CNS) axons, injury causes a total failure to regenerate. This failure is due to both the intrinsic developmental decrease in growth capacity and the extrinsic inhibitory environment formed because of the injury. One way to re-invigorate mature axons into a regrowth state is to induce regenerative gene expression in the nucleus to increase the intrinsic growth state of the neuron. One potential mechanism is through changes in epigenetic factors. Another possible method is to alter posttranslational modifications in axonal and growth cone microtubules of the axonal cytoplasm (Trakhtenberg and Goldberg, 2012; Cho and Cavalli, 2014). An increasing number of studies have turned to epigenetic manipulation using pharmacological inhibitors to try and enhance axonal growth. Numerous reversible posttranslational modifications are considered important for axonal growth and regeneration but such modifications are regulated by families of enzymes which affect system wide changes in the body, sometimes shuttling between the nucleus and the cytoplasm as well as between the cell body and axon. Two families of enzymes, histone acetyl transferases (HATs) and histone deacetylases (HDACs), act antagonistically to affect acetylation and de-acetylation respectively on many proteins throughout the body. Acetylation in neurons is critical for many physiological events such as cell division, cell growth and proliferation. This paper will focus on the potential implications of acetylation homeostasis on axonal growth.

Integrating the Alzheimer's Disease Proteome and Transcriptome: A Comprehensive Network Model of a Complex Disease

Network models combined with gene expression studies have become useful tools for studying complex diseases like Alzheimer's disease. We constructed a "Core" Alzheimer's disease protein interaction network by human curation of the primary literature. The Core network consisted of 775 nodes and 2,204 interactions. To our knowledge, this is the most comprehensive and accurate protein interaction network yet constructed for Alzheimer's disease. An "Expanded" network was computationally constructed by adding additional proteins that interacted with Core network proteins, and consisted of 4,945 nodes and 26,064 interactions. We then mapped existing gene expression studies to the Core network. This combined data model identified the MAPK/ERK pathway and clathrin-mediated receptor endocytosis as key pathways in Alzheimer's disease. Important proteins in the MAPK/ERK pathway that interacted in the Core network formed a downregulated cluster of nodes, whereas clathrin and several clathrin accessory proteins that interacted in the Core network formed an upregulated cluster of nodes. The MAPK/ERK pathway is a key component in synaptic plasticity and learning, processes disrupted in Alzheimer's. Clathrin and clathrin adaptor proteins are involved in the endocytosis of the APP protein that can lead to increased intracellular levels of amyloid beta peptide, contributing to the progression of Alzheimer's.

Drosophila melanogaster gene Merlin interacts with the clathrin adaptor protein gene lap

The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer(DBB) together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.

Dynamin 2 mutations associated with human diseases impair clathrin-mediated receptor endocytosis

Dynamin 2 (DNM2) is a large GTPase involved in the release of nascent vesicles during endocytosis and intracellular membrane trafficking. Distinct DNM2 mutations, affecting the middle domain (MD) and the Pleckstrin homology domain (PH), have been identified in autosomal dominant centronuclear myopathy (CNM) and in the intermediate and axonal forms of the Charcot-Marie-Tooth peripheral neuropathy (CMT). We report here the first CNM mutation (c.1948G>A, p.E650 K) in the DNM2 GTPase effector domain (GED), leading to a slowly progressive moderate myopathy. COS7 cells transfected with DNM2 constructs harboring a disease-associated mutation in MD, PH, or GED show a reduced uptake of transferrin and low-density lipoprotein (LDL) complex, two markers of clathrin-mediated receptor endocytosis. A decrease in clathrin-mediated endocytosis was also identified in skin fibroblasts from one CNM patient. We studied the impact of DNM2 mutant overexpression on epidermal growth factor (EGF)-induced extracellular signal-regulated kinase 1 (ERK1) and ERK2 activation, known to be an endocytosis- and DNM2-dependent process. Activation of ERK1/2 was impaired for all the transfected mutants in COS7 cells, but not in CNM fibroblasts. Our results indicate that impairment of clathrin-mediated endocytosis may play a role in the pathophysiological mechanisms leading to DNM2-related diseases, but the tissue-specific impact of DNM2 mutations in both diseases remains unclear.

Calpain Hydrolysis of α- and β2-Adaptins Decreases Clathrin-dependent Endocytosis and May Promote Neurodegeneration

Clathrin-dependent endocytosis is mediated by a tightly regulated network of molecular interactions that provides essential protein-protein and protein-lipid binding activities. Here we report the hydrolysis of the alpha- and beta2-subunits of the tetrameric adaptor protein complex 2 by calpain. Calcium-dependent alpha- and beta2-adaptin hydrolysis was observed in several rat tissues, including brain and primary neuronal cultures. Neuronal alpha- and beta2-adaptin cleavage was inducible by glutamate stimulation and was accompanied by the decreased endocytosis of transferrin. Heterologous expression of truncated forms of the beta2-adaptin subunit significantly decreased the membrane recruitment of clathrin and inhibited clathrin-mediated receptor endocytosis. Moreover, the presence of truncated beta2-adaptin sensitized neurons to glutamate receptor-mediated excitotoxicity. Proteolysis of alpha- and beta2-adaptins, as well as the accessory clathrin adaptors epsin 1, adaptor protein 180, and the clathrin assembly lymphoid myeloid leukemia protein, was detected in brain tissues after experimentally induced ischemia and in cases of human Alzheimer disease. The present study further clarifies the central role of calpain in regulating clathrin-dependent endocytosis and provides evidence for a novel mechanism through which calpain activation may promote neurodegeneration: the sensitization of cells to glutamate-mediated excitotoxicity via the decreased internalization of surface receptors.

Mechanisms of Short-Term Desensitization of GIRK Channel Activity

G protein-coupled receptors (GPCR) signaling is precisely timed. This can be achieved at the level of the G protein activation and/or at the receptor level. At the receptor level, classical desensitization is believed to be controlled by a decrease in receptors number in the membrane: Activated receptors are phosphorylated by GPCR-kinase (GRK), tagging them for arrestin binding. This in-turn leads to clathrin-mediated receptor endocytosis. This multisteps process is extended in time (min), and is dependent on initial GPCR phosphorylation by GRK. In contrast, effector regulation is controlled at much faster time scale (s). Here we used the G protein-coupled inwardly rectifying K+ channels (GIRK), as an effector model for GPCR activation, to achieve real-time readout, in intact cells, of GPCR-mediated effector regulation. Upon GIRK activation, currents were desensitized within seconds. This regulation may serve as fast negative feedback for GPCR activation. Here we show that GIRK desensitization rates are dependent on GRK2 activity. GRK2 accelerates desensitization of currents induced by some Gαi/o coupled receptors, but not all. In contrast, silencing endogenous GRK2, results in a decreased in the desensitization rates. A dominant-negative mutant lacking kinase activity did not affect the ability of GRK2 to accelerate currents desensitization, suggesting the lack of GPCR classical desensitization in this process. In contrast, GRK2 mutant displaying limited Gβγ binding affinity, failed to accelerate current desensitization.Measuring simultaneously membrane fluorescence signals from cells expressing GFP-GRK2 and GIRK activity under TIRF microscopy and electrophysiological recordings, respectively, show that GFP-GRK2 membrane recruitment and GIRK desensitization occurs simultaneously.We suggest that GPCR activation induces GRK2 recruitment to the membrane, where it competes with GIRK for Gβγ, to result in short-term current desensitization. The results will be discussed in terms of a novel mechanism of selective regulation of effectors by GPCRs.

A Conserved Cys-loop Receptor Aspartate Residue in the M3-M4 Cytoplasmic Loop Is Required for GABAA Receptor Assembly

Members of the Cys-loop superfamily of ligand-gated ion channels, which mediate fast synaptic transmission in the nervous system, are assembled as heteropentamers from a large repertoire of neuronal subunits. Although several motifs in subunit N-terminal domains are known to be important for subunit assembly, increasing evidence points toward a role for C-terminal domains. Using a combination of flow cytometry, patch clamp recording, endoglycosidase H digestion, brefeldin A treatment, and analytic centrifugation, we identified a highly conserved aspartate residue at the boundary of the M3-M4 loop and the M4 domain that was required for binary and ternary gamma-aminobutyric acid type A receptor surface expression. Mutation of this residue caused mutant and partnering subunits to be retained in the endoplasmic reticulum, reflecting impaired forward trafficking. Interestingly although mutant and partnering wild type subunits could be coimmunoprecipitated, analytic centrifugation studies demonstrated decreased formation of pentameric receptors, suggesting that this residue played an important role in later steps of subunit oligomerization. We thus conclude that C-terminal motifs are also important determinants of Cys-loop receptor assembly.

Interactions between the Three CIN85 SH3 Domains and Ubiquitin: Implications for CIN85 Ubiquitination

CIN85 is an adaptor protein linking the ubiquitin ligase Cbl and clathrin-binding proteins in clathrin-mediated receptor endocytosis. The SH3 domains of CIN85 bind to a proline-rich region of Cbl. Here we show that all three SH3 domains of CIN85 bind to ubiquitin. We also present a data-based structural model of the CIN85 SH3-C domain in complex with ubiquitin. In this complex, ubiquitin binds to the canonical interaction surface of the SH3 domain for proline-rich ligands and mimics the PPII helix, and we provide evidence that ubiquitin competes with these ligands for binding. We demonstrate that disruption of ubiquitin binding results in constitutive ubiquitination of CIN85 and an increased level of ubiquitination of EGFR in the absence of EGF stimulation. These results suggest that competition between Cbl and ubiquitin binding to CIN85 regulates Cbl function and EGFR endocytosis.

Ischemic Insults Direct Glutamate Receptor Subunit 2-Lacking AMPA Receptors to Synaptic Sites

Regulated AMPA receptor (AMPAR) trafficking at excitatory synapses is a mechanism critical to activity-dependent alterations in synaptic efficacy. The role of regulated AMPAR trafficking in insult-induced synaptic remodeling and/or cell death is, however, as yet unclear. Here we show that brief oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia, promotes redistribution of AMPARs at synapses of hippocampal neurons, leading to a switch in AMPAR subunit composition. Ischemic insults promote internalization of glutamate receptor subunit 2 (GluR2)-containing AMPARs from synaptic sites via clathrin-dependent endocytosis and facilitate delivery of GluR2-lacking AMPARs to synaptic sites via soluble N-ethylmaleimide-sensitive factor attachment protein receptor-dependent exocytosis, evident at early times after insult. The OGD-induced switch in receptor subunit composition requires PKC activation, dissociation of GluR2 from AMPA receptor-binding protein, and association with protein interacting with C kinase-1. We further show that AMPARs at synapses of insulted neurons exhibit functional properties of GluR2-lacking AMPARs. AMPAR-mediated miniature EPSCs exhibit increased amplitudes and enhanced sensitivity to subunit-specific blockers of GluR2-lacking AMPARs, evident at 24 h after ischemia. The OGD-induced alterations in synaptic AMPA currents require clathrin-mediated receptor endocytosis and PKC activation. Thus, ischemic insults promote targeting of GluR2-lacking AMPARs to synapses of hippocampal neurons, mechanisms that may be relevant to ischemia-induced synaptic remodeling and/or neuronal death.

Distinct β-Arrestin- and G Protein-dependent Pathways for Parathyroid Hormone Receptor-stimulated ERK1/2 Activation

Parathyroid hormone (PTH) regulates calcium homeostasis via the type I PTH/PTH-related peptide (PTH/PTHrP) receptor (PTH1R). The purpose of the present study was to identify the contributions of distinct signaling mechanisms to PTH-stimulated activation of the mitogen-activated protein kinases (MAPK) ERK1/2. In Human embryonic kidney 293 (HEK293) cells transiently transfected with hPTH1R, PTH stimulated a robust increase in ERK activity. The time course of ERK1/2 activation was biphasic with an early peak at 10 min and a later sustained ERK1/2 activation persisting for greater than 60 min. Pretreatment of HEK293 cells with the PKA inhibitor H89 or the PKC inhibitor GF109203X, individually or in combination reduced the early component of PTH-stimulated ERK activity. However, these inhibitors of second messenger dependent kinases had little effect on the later phase of PTH-stimulated ERK1/2 phosphorylation. This later phase of ERK1/2 activation at 30-60 min was blocked by depletion of cellular beta-arrestin 2 and beta-arrestin 1 by small interfering RNA. Furthermore, stimulation of hPTH1R with PTH analogues, [Trp1]PTHrp-(1-36) and [d-Trp12,Tyr34]PTH-(7-34), selectively activated G(s)/PKA-mediated ERK1/2 activation or G protein-independent/beta-arrestin-dependent ERK1/2 activation, respectively. It is concluded that PTH stimulates ERK1/2 through several distinct signal transduction pathways: an early G protein-dependent pathway meditated by PKA and PKC and a late pathway independent of G proteins mediated through beta-arrestins. These findings imply the existence of distinct active conformations of the hPTH1R responsible for the two pathways, which can be stimulated by unique ligands. Such ligands may have distinct and valuable therapeutic properties.

Ubiquitylation and cell signaling

Ubiquitylation is an emerging mechanism implicated in a variety of nonproteolytic cellular functions. The attachment of a single ubiquitin (Ub) or poly-Ub (lysine 63) chains to proteins control gene transcription, DNA repair and replication, intracellular trafficking and virus budding. In these processes, protein ubiquitylation exhibits inducibility, reversibility and recognition by specialized domains, features similar to protein phosphorylation, which enable Ub to act as a signaling device. Here, we highlight several recent examples on how Ub regulates signaling and how signaling regulates ubiquitylation during physiological and pathological cellular processes.

CPG2

Long-term maintenance and modification of synaptic strength involve the turnover of neurotransmitter receptors. Glutamate receptors are constitutively and acutely internalized, presumptively through clathrin-mediated receptor endocytosis. Here, we show that cpg2 is a brain-specific splice variant of the syne-1 gene that encodes a protein specifically localized to a postsynaptic endocytotic zone of excitatory synapses. RNAi-mediated CPG2 knockdown increases the number of postsynaptic clathrin-coated vesicles, some of which traffic NMDA receptors, disrupts the constitutive internalization of glutamate receptors, and inhibits the activity-induced internalization of synaptic AMPA receptors. Manipulating CPG2 levels also affects dendritic spine size, further supporting a function in regulating membrane transport. Our results suggest that CPG2 is a key component of a specialized postsynaptic endocytic mechanism devoted to the internalization of synaptic proteins, including glutamate receptors. The activity dependence and distribution of cpg2 expression further suggest that it contributes to the capacity for postsynaptic plasticity inherent to excitatory synapses.

The Adaptor Complex 2 Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Endocytosis

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

A G Protein-coupled Receptor Kinase Induces XenopusOocyte Maturation

Several recent studies have suggested that resumption of oocyte meiosis, indicated by germinal vesicle breakdown or GVBD, involves inhibition of endogenous heterotrimeric G proteins in both frogs and mice. These studies imply that a heterotrimeric G protein(s), and hence its upstream activator (a G protein-coupled receptor or GpCR), is activated in prophase oocytes and is responsible for maintaining meiosis arrest. To test the existence and function of this putative GpCR, we utilized a mammalian G-protein-coupled receptor kinase (GRK3) and beta-arrestin-2, which together are known to cause GpCR desensitization. Injection of mRNA for rat GRK3 caused hormone-independent GVBD. The kinase activity of GRK3 was essential for GVBD induction as its kinase-dead mutant (GRK3-K220R) was completely ineffective. Another GRK3 mutant (GRK3-DeltaC), which lacked the C-terminal G(betagamma)-binding domain and which was not associated with oocyte membranes, also failed to induce GVBD. Furthermore, injection of rat beta-arrestin-2 mRNA also induced hormone-independent GVBD. Several inhibitors of clathrin-mediated receptor endocytosis (the clathrin-binding domain of beta-arrestin-2, concanavalin A, and monodansyl cadaverine) significantly reduced the abilities of GRK3/beta-arrestin-2 to induce GVBD. These results support the central role of a yet-unidentified GpCR in maintaining prophase arrest in frog oocytes and provide a potential means for its molecular identification.