Number Of Surface Receptors Research Articles

PKCε regulates GABA A receptor trafficking through NSF

GABAA receptors, the major mediators of fast synaptic inhibition in the brain, are composed primarily of α, β, and γ 2 subunits. Much evidence indicates that activation of protein kinase C (PKC) stimulates GABAA receptor trafficking, causing a decrease in the density of cell surface receptors. The PKC isozymes that mediate these responses are not known. Using human embryonic kidney cells (A293) heterologously expressing α 1 β 2γ 2S receptors and a PKCε analog-sensitive mutant, we found that selective inhibition of PKCε prevents phorbol ester-stimulated decreases in the number of cell surface receptors. Immunoprecipitation studies revealed that PKCε associates in a complex with the receptor and with NSF (N-ethylmaleimide-sensitive factor), an ATPase that regulates receptor trafficking. The association between PKCε and NSF but not PKCε and γ 2 subunits was dependent on the kinase activity of PKCε as it was blocked by a PKCε inhibitor and enhanced by phorbol ester. Pull-down and overlay studies revealed that PKCε and NSF bind directly to each other. Immunofluorescence studies showed that NSF colocalizes with PKCε and γ 2 subunits at the perinuclear cytoplasm in HEK293 and cerebellar granular cells and that phorbol ester treatment caused both to translocate to the cell surface. Translocation of NSF was prevented by a PKCε inhibitor. PKCε phosphorylated NSF in vitro. These results suggest a molecular mechanism whereby PKCε regulates the trafficking GABAA receptors through phosphorylation and translocation of NSF. Supported by PHS grant AA013588 and by funds provided by the State of California for medical research on alcohol and substance abuse through UCSF.

Luteinizing hormone receptor ectodomain splice variant misroutes the full-length receptor into a subcompartment of the endoplasmic reticulum.

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor that is expressed in multiple RNA messenger forms. The common rat ectodomain splice variant is expressed concomitantly with the full-length LHR in tissues and is a truncated transcript corresponding to the partial ectodomain with a unique C-terminal end. Here we demonstrate that the variant alters the behavior of the full-length receptor by misrouting it away from the normal secretory pathway in human embryonic kidney 293 cells. The variant was expressed as two soluble forms of M(r) 52,000 and M(r) 54,000, but although the protein contains a cleavable signal sequence, no secretion to the medium was observed. Only a very small fraction of the protein was able to gain hormone-binding ability, suggesting that it is retained in the endoplasmic reticulum (ER) by its quality control due to misfolding. This was supported by the finding that the variant was found to interact with calnexin and calreticulin and accumulated together with these ER chaperones in a specialized juxtanuclear subcompartment of the ER. Only proteasomal blockade with lactacystin led to accumulation of the variant in the cytosol. Importantly, coexpression of the variant with the full-length LHR resulted in reduction in the number of receptors that were capable of hormone binding and were expressed at the cell surface and in targeting of immature receptors to the juxtanuclear ER subcompartment. Thus, the variant mediated misrouting of the newly synthesized full-length LHRs may provide a way to regulate the number of cell surface receptors.

Induction of mitochondrial ROS production by electrophilic lipids: a new pathway of redox signaling?

reactive oxygen species (ROS), such as superoxide or hydrogen peroxide, are usually thought of as damaging agents that nonspecifically disrupt proteins, lipids, and nucleic acids ([1][1]). However, there is now considerable evidence that ROS also act as signaling agents in a range of biological

Receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis

The anthrax toxin is composed of three independent polypeptide chains. Successful intoxication only occurs when heptamerization of the receptor-binding polypeptide, the protective antigen (PA), allows binding of the two enzymatic subunits before endocytosis. We show that this tailored behavior is caused by two counteracting posttranslational modifications in the cytoplasmic tail of PA receptors. The receptor is palmitoylated, and this unexpectedly prevents its association with lipid rafts and, thus, its premature ubiquitination. This second modification, which is mediated by the E3 ubiquitin ligase Cbl, only occurs in rafts and is required for rapid endocytosis of the receptor. As a consequence, cells expressing palmitoylation-defective mutant receptors are less sensitive to anthrax toxin because of a lower number of surface receptors as well as premature internalization of PA without a requirement for heptamerization.

Antibody-based therapeutics: Focus on prostate cancer

The recent clinical and commercial success of anti-cancer antibodies such as rituximab, trastuzumab, cetuximab and bevacizumab has continued to foster great interest in antibody-based therapeutics for the treatment of both hematopoietic malignancies and solid tumors. Given the likely lower toxicity for antibodies which, in contrast with traditional cytotoxic small molecule drugs, target tumor cells and have a lower impact on non-malignant by-stander organs, the potential increases in efficacy associated with conjugation to radioisotopes and other cellular toxins and the ability to characterize the target with clinical laboratory diagnostics to improve the drugs clinical performance, it is anticipated that current and future antibody therapeutics will find substantial roles alone and in combination therapy strategies for the treatment of patients with cancer. A significant number of cell surface proteins, glycoproteins, receptors, enzymes and peptides have been discovered that have become targets for the treatment of advanced hormone-refractory prostate cancer. A variety of naked antibodies and antibody conjugates have currently progressed through preclinical development and are in early or more advanced stages of clinical development. Clinicians, scientists and prostate cancer patients are all keenly interested to learn whether these agents when administered alone or in combination with other hormonal-based and cytotoxic therapies will show lasting benefit for sufferers of this common disease.

Functional Properties of Internalization-Deficient P2X4 Receptors Reveal a Novel Mechanism of Ligand-Gated Channel Facilitation by Ivermectin

Although P2X receptors within the central nervous system mediate excitatory ATP synaptic transmission, the identity of central ATP-gated channels has not yet been elucidated. P2X(4), the most widely expressed subunit in the brain, was previously shown to undergo clathrin-dependent constitutive internalization by direct interaction between activator protein (AP)2 adaptors and a tyrosine-based sorting signal specifically present in the cytosolic C-terminal tail of mammalian P2X(4) sequences. In this study, we first used internalization-deficient P2X(4) receptor mutants to show that suppression of the endocytosis motif significantly increased the apparent sensitivity to ATP and the ionic permeability of P2X(4) channels. These unique properties, observed at low channel density, suggest that interactions with AP2 complexes may modulate the function of P2X(4) receptors. In addition, ivermectin, an allosteric modulator of several receptor channels, including mammalian P2X(4), did not potentiate the maximal current of internalization-deficient rat or human P2X(4) receptors. We demonstrated that binding of ivermectin onto wild-type P2X(4) channels increased the fraction of plasma membrane P2X(4) receptors, whereas surface expression of internalization-deficient P2X(4) receptors remained unchanged. Disruption of the clathrin-mediated endocytosis with the dominant-negative mutants Eps15 or AP-50 abolished the ivermectin potentiation of wild-type P2X(4) channel currents. Likewise, ivermectin increased the membrane fraction of nicotinic alpha7 acetylcholine (nalpha7ACh) receptors and the potentiation of acetylcholine current by ivermectin was suppressed when the same dominant-negative mutants were expressed. These data showed that potentiation by ivermectin of both P2X(4) and nalpha7ACh receptors was primarily caused by an increase in the number of cell surface receptors resulting from a mechanism dependent on clathrin/AP2-mediated endocytosis.

Ubiquilin-1 Regulates Nicotine-induced Up-regulation of Neuronal Nicotinic Acetylcholine Receptors

Chronic exposure to nicotine, as in tobacco smoking, up-regulates nicotinic acetylcholine receptor surface expression in neurons. This up-regulation has been proposed to play a role in nicotine addiction and withdrawal. The regulatory mechanisms behind nicotine-induced up-regulation of surface nicotinic acetylcholine receptors remain to be determined. It has recently been suggested that nicotine stimulation acts through increased assembly and maturation of receptor subunits into functional pentameric receptors. Studies of muscle nicotinic acetylcholine receptors suggest that the availability of unassembled subunits in the endoplasmic reticulum can be regulated by the ubiquitin-proteosome pathway, resulting in altered surface expression. Here, we describe a role for ubiquilin-1, a ubiquitin-like protein with the capacity to interact with both the proteosome and ubiquitin ligases, in regulating nicotine-induced up-regulation of neuronal nicotinic acetylcholine receptors. Ubiquilin-1 interacts with unassembled alpha3 and alpha4 subunits when coexpressed in heterologous cells and interacts with endogenous nicotinic acetylcholine receptors in neurons. Coexpression of ubiquilin-1 and neuronal nicotinic acetylcholine receptors in heterologous cells dramatically reduces the expression of the receptors on the cell surface. In cultured superior cervical ganglion neurons, expression of ubiquilin-1 abolishes nicotine-induced up-regulation of nicotinic acetylcholine receptors but has no effect on the basal level of surface receptors. Coimmunostaining shows that the interaction of ubiquilin-1 with the alpha3 subunit draws the receptor subunit and proteosome into a complex. These data suggest that ubiquilin-1 limits the availability of unassembled nicotinic acetylcholine receptor subunits in neurons by drawing them to the proteosome, thus regulating nicotine-induced up-regulation.

Phospho-dependent binding of the clathrin AP2 adaptor complex to GABA A receptors regulates the efficacy of inhibitory synaptic transmission

The efficacy of synaptic inhibition depends on the number of gamma-aminobutyric acid type A receptors (GABA(A)Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABA(A)R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA(A)R beta subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the beta3 subunit) incorporates the major sites of serine phosphorylation within receptor beta subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepbeta3) corresponding to the AP2 binding motif in the GABA(A)R beta3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepbeta3 peptide, but not its phosphorylated equivalent (pepbeta3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA(A)R current. These effects of pepbeta3 on GABA(A)R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepbeta3 on GABA(A)R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA(A)Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.

Subtype-selective Interaction with the Transcription Factor CCAAT/Enhancer-binding Protein (C/EBP) Homologous Protein (CHOP) Regulates Cell Surface Expression of GABAB Receptors

The metabotropic gamma-aminobutyric acid, type B (GABA(B)) receptors mediate the slow component of GABAergic transmission in the brain. Functional GABA(B) receptors are heterodimers of the two subunits GABA(B1) and GABA(B2), of which GABA(B1) exists in two main isoforms, GABA(B1a) and GABA(B1b). The significance of the structural heterogeneity of GABA(B) receptors, the mechanism leading to their differential targeting in neurons as well as the regulation of cell surface numbers of GABA(B) receptors, is poorly understood. To gain insights into these processes, we searched for proteins interacting with the C-terminal domain of GABA(B2). Here, we showed that the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) directly interacts with GABA(B) receptors in a subtype-selective manner to regulate cell surface expression of GABA(B1a)/GABA(B2) receptors upon co-expression in HEK 293 cells. The interaction of CHOP with GABA(B1a)/GABA(B2) receptors resulted in their intracellular accumulation and in a reduced number of cell surface receptors. This regulation required the interaction of CHOP via two distinct domains with the heterodimeric receptor; its C-terminal leucine zipper associates with the leucine zipper present in the C-terminal domain of GABA(B2), and its N-terminal domain associates with an as yet unidentified site on GABA(B1a). In conclusion, the data indicated a subtype-selective regulation of cell surface receptors by interaction with the transcription factor CHOP.

Chronic Nicotine Exposure Upregulates Nicotinic Receptors by a Novel Mechanism

Nicotine addiction is initiated by its binding to high-affinity nicotinic receptors in brain composed primarily of alpha4 and beta2 subunits. For nicotinic receptors expressed in vivo or heterologously, nicotine exposure over hours to days increases or "upregulates" high-affinity nicotine binding to receptors through a posttranslational mechanism thought to increase receptor numbers. Using heterologous expression, we find nicotine exposure causes a fourfold to sixfold higher binding to alpha4beta2 receptors that does not correspond with any significant change in the number of surface receptors or a change in the assembly, trafficking, or cell-surface turnover of the receptors. However, upregulation does alter the functional state of the receptor, slowing desensitization and enhancing sensitivity to acetylcholine. Based on these findings, we propose an alternative mechanism to explain nicotine-induced upregulation in which nicotine exposure slowly stabilizes alpha4beta2 receptors in a high-affinity state that is more easily activated, thereby providing a memory for nicotine exposure.

Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of α4β2 Nicotinic Acetylcholine Receptors

The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed alpha(4)beta(2) nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested.

CART: An Hrs/Actinin-4/BERP/Myosin V Protein Complex Required for Efficient Receptor Recycling

Altering the number of surface receptors can rapidly modulate cellular responses to extracellular signals. Some receptors, like the transferrin receptor (TfR), are constitutively internalized and recycled to the plasma membrane. Other receptors, like the epidermal growth factor receptor (EGFR), are internalized after ligand binding and then ultimately degraded in the lysosome. Routing internalized receptors to different destinations suggests that distinct molecular mechanisms may direct their movement. Here, we report that the endosome-associated protein hrs is a subunit of a protein complex containing actinin-4, BERP, and myosin V that is necessary for efficient TfR recycling but not for EGFR degradation. The hrs/actinin-4/BERP/myosin V (CART [cytoskeleton-associated recycling or transport]) complex assembles in a linear manner and interrupting binding of any member to its neighbor produces an inhibition of transferrin recycling rate. Disrupting the CART complex results in shunting receptors to a slower recycling pathway that involves the recycling endosome. The novel CART complex may provide a molecular mechanism for the actin-dependence of rapid recycling of constitutively recycled plasma membrane receptors.

Rapid Activity-Driven SNARE-Dependent Trafficking of Nicotinic Receptors on Somatic Spines

Rapid trafficking of glutamate receptors contributes importantly to synaptic plasticity, but whether similar trafficking extends to other ionotropic receptors is unknown. Nicotinic acetylcholine receptors containing alpha7 subunits are widely expressed in the nervous system and allow calcium influx. Because of this, alpha7-containing receptors regulate diverse events, depending on the signaling pathways available. We find that the receptors codistribute with target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) postsynaptically and that nicotinic stimulation rapidly induces SNARE-dependent vesicular endocytosis accompanied by receptor internalization. At the same time, a SNARE-dependent process recruits receptors to the cell surface from internal pools. Overall, the trafficking does not markedly change the number of surface receptors or their combined whole-cell response to nicotine. SNARE-dependent trafficking is needed, however, for the receptors to remain capable of activating the transcription factor cAMP response element-binding protein and attendant gene expression when repeatedly challenged. Thus, trafficking appears to be essential for maintaining functional coupling between alpha7-receptor responses and downstream signaling.

Natural killer cells and cross‐talk with dendritic cells

SummaryNatural Killer (NK) cells, a major actor of the innate immunity, play a central role in defences against viral infectious and tumor growth. NK cells are regulated by a number of surface receptors that either activate or inhibit their function. Of major interest are the inhibitory receptors specific for HLA‐class I molecules. These receptors allow to discriminate between normal cells, expressing sufficient amounts of HLA‐class I molecules, and cells that have lost the surface expression of one or more HLA‐class I alleles, as it occurs in virus‐infected or tumor cells. As shown by recent studies, NK cells can interact with dendritic cells (DC), another important player of the innate immunity. The cross‐talk between these cells, taking place in inflamed tissues and lymph nodes, may shape the functional activities of both cell types and influence downstream adaptive responses initiated by the interaction between DC and T lymphocyte in secondary lymphoid tissues.

Structured model of influenza virus replication in MDCK cells.

Intracellular events that take place during influenza virus replication in animal cells are well understood qualitatively. However, to better understand the complex interaction of the virus with its host cell and to quantitatively analyze the use of cellular resources for virion formation or the overall dynamic for the entire infection cycle, a mathematical model for influenza virus replication has to be formulated. Here, we present a structured model for the single-cell reproductive cycle of influenza A virus in animal cells that accounts for the individual steps of the process such as attachment, internalization, genome replication and translation, and progeny virion assembly. The model describes an average cell surrounded by a small quantity of medium and infected by a low number of virus particles. The model allows estimation of the cellular resources consumed by virus replication. Simulation results show that the number of cellular surface receptors and endosomes, as well as other resources, such as the number of free nucleotides or amino acids, is not significantly influenced by influenza virus propagation. A factor that limits the growth rate of progeny viruses and their release is the total amount of matrix proteins (M1) in the nucleus while other newly synthesized viral proteins (e.g., nucleoprotein NP) and viral RNAs accumulate. During budding, synthesis of vRNPs (viral ribonucleoprotein complexes) represents another limiting factor. Based on this model it is also possible to analyze effects of parameter changes on the dynamics of virus replication, to identify possible targets for molecular engineering, or to develop strategies for improving yields in vaccine production processes. Furthermore, a better insight into the interactions of viruses and host cells might help to improve our understanding of virus-related diseases and to develop therapies.

Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating gamma-aminobutyric acid type A receptor membrane trafficking.

Gamma-aminobutyric acid type A receptors (GABA(A)Rs) are the major sites of fast synaptic inhibition in the brain. An essential determinant for the efficacy of synaptic inhibition is the regulation of GABA(A)R cell surface stability. Here, we have examined the regulation of GABA(A)R endocytic sorting, a critical regulator of cell surface receptor number. In neurons, rapid constitutive endocytosis of GABA(A)Rs was evident. Internalized receptors were then either rapidly recycled back to the cell surface, or on a slower time scale, targeted for lysosomal degradation. This sorting decision was regulated by a direct interaction of GABA(A)Rs with Huntingtin-associated protein 1 (HAP1). HAP1 modulated synaptic GABA(A)R number by inhibiting receptor degradation and facilitating receptor recycling. Together these observations have identified a role for HAP1 in regulating GABA(A)R sorting, suggesting an important role for this protein in the construction and maintenance of inhibitory synapses.

Palmitoylation regulates the clustering and cell surface stability of GABA A receptors

GABA A receptors are the major mediators of fast synaptic inhibition in the brain. These receptors are ionotropic, hetero-pentameric, ligand-gated ion channels, which are predominantly composed of α, β, and γ2 subunits. Here, we reveal that the γ2 subunit of neuronal and recombinant GABA A receptors is palmitoylated. We further establish that palymitoylation of the γ2 subunit occurs on multiple cysteine residues within the major intracellular domain of this receptor subunit. In cultured hippocampal neurons, inhibitors of protein palymitoylation reduced the synaptic clustering of GABA A receptors and steady-state cell surface receptor number. These effects are likely to be mediated by direct palmitoylation of the γ2 subunit, as mutation of palmitoylation sites within this protein reduces GABA A receptor clustering. Taken together, these results suggest that palmitoylation of GABA A receptors plays an essential role in regulating the clustering of these receptors at synaptic sites.

Comparative usage of herpesvirus entry mediator A and nectin-1 by laboratory strains and clinical isolates of herpes simplex virus

The herpesvirus entry mediator A (HVEM/HveA) and nectin-1 (HveC/CD111) are two major receptors for herpes simplex virus (HSV). Although structurally unrelated, both receptors can independently mediate entry of wild-type (wt) HSV-1 and HSV-2 by interacting with the viral envelope glycoprotein D (gD). Laboratory strains with defined mutations in gD (e.g. rid1) do not use HVEM but use nectin-2 (HveB/CD112) for entry. The relative usage of HVEM and nectin-1 during HSV infection in vivo is not known. In the absence of a defined in vivo model, we used in vitro approaches to address this question. First, we screened HSV clinical isolates from various origins for receptor tropism and found that all used both HVEM and nectin-1. Second, we determined the numbers of surface receptors on various susceptible and resistant cell lines as well as on primary fibroblasts derived from an individual with cleft lip/palate ectodermal dysplasia (CLPED1). Although CLPED1 cells can only express a defective form of nectin-1, they allowed entry of wild type and mutant HSV strains by usage of either HVEM or nectin-2. Finally, we compared the ability of HVEM and nectin-1 to mediate entry when expressed at varying cell surface densities. Both receptors showed a direct relationship between the number of receptors and HSV susceptibility. Direct comparison of receptors suggests that nectin-1 is more efficient at promoting entry than HVEM. Overall, our data suggest that both receptors play a role during HSV infection in vivo and that both are highly efficient even at low levels of expression.

Phosphorylation and Chronic Agonist Treatment Atypically Modulate GABAB Receptor Cell Surface Stability

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.

Mechanisms of E3 modulation of immune and inflammatory responses.

Adenoviruses contain genes that have evolved to control the host immune and inflammatory responses; however, it is not clear whether these genes function primarily to facilitate survival of the virus during acute infection or during its persistent phase. These issues have assumed greater importance as the use of adenoviruses as vectors for gene therapy has been expanded. This review will focus on the mechanism of immune evasion mediated by the proteins encoded within the early region 3 (E3) transcription region, which affect the functions of a number of cell surface receptors including Fas, intracellular cell signaling events involving NF-kappaB, and the secretion of pro-inflammatory molecules such as chemokines. The successful use of E3 genes in facilitating allogeneic transplantation and in preventing autoimmune diabetes in several transgenic mouse models will also be described.