Regulation of AMPA Receptor Trafficking and Function by Glycogen Synthase Kinase 3

Accumulating evidence suggests that glycogen synthase kinase 3 (GSK-3) is a multifunctional kinase implicated in neuronal development, mood stabilization, and neurodegeneration. However, the synaptic actions of GSK-3 are largely unknown. In this study, we examined the impact of GSK-3 on AMPA receptor (AMPAR) channels, the major mediator of excitatory transmission, in cortical neurons. Application of GSK-3 inhibitors or knockdown of GSK-3 caused a significant reduction of the amplitude of miniature excitatory postsynaptic current (mEPSC), a readout of the unitary strength of synaptic AMPARs. Treatment with GSK-3 inhibitors also decreased surface and synaptic GluR1 clusters on dendrites and increased internalized GluR1 in cortical cultures. Rab5, the small GTPase controlling the transport from plasma membrane to early endosomes, was activated by GSK-3 inhibitors. Knockdown of Rab5 prevented GSK-3 inhibitors from regulating mEPSC amplitude. Guanyl nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab5 between membrane and cytosol, formed an increased complex with Rab5 after treatment with GSK-3 inhibitors. Blocking the function of GDI occluded the effect of GSK-3 inhibitors on mEPSC amplitude. In cells transfected with the non-phosphorylatable GDI mutant, GDI(S45A), GSK-3 inhibitors lost the capability to regulate GDI-Rab5 complex, mEPSC amplitude, and AMPAR surface expression. These results suggest that GSK-3, via altering the GDI-Rab5 complex, regulates Rab5-mediated endocytosis of AMPARs. It provides a potential mechanism underlying the role of GSK-3 in synaptic transmission and plasticity.     

Differential Regulation of GABAB Receptor Trafficking by Different Modes of N-methyl-d-aspartate (NMDA) Receptor Signaling

Inhibitory GABA_B receptors (GABA_BRs) can down-regulate most excitatory synapses in the CNS by reducing postsynaptic excitability. Functional GABA_BRs are heterodimers of GABA_B1 and GABA_B2 subunits and here we show that the trafficking and surface expression of GABA_BRs is differentially regulated by synaptic or pathophysiological activation of NMDA receptors (NMDARs). Activation of synaptic NMDARs using a chemLTP protocol increases GABA_BR recycling and surface expression. In contrast, excitotoxic global activation of synaptic and extrasynaptic NMDARs by bath application of NMDA causes the loss of surface GABA_BRs. Intriguingly, exposing neurons to extreme metabolic stress using oxygen/glucose deprivation (OGD) increases GABA_B1 but decreases GABA_B2 surface expression. The increase in surface GABA_B1 involves enhanced recycling and is blocked by the NMDAR antagonist AP5. The decrease in surface GABA_B2 is also blocked by AP5 and by inhibiting degradation pathways. These results indicate that NMDAR activity is critical in GABA_BR trafficking and function and that the individual subunits can be separately controlled to regulate neuronal responsiveness and survival.     

Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking

Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated after endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules with the use of reversibly biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. With the use of a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that after internalization, EGFR remained active in the early endosomes. However, receptors were inactivated before degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, whereas others, such as Eps8, were found only with intracellular receptors. During the inactivation phase, c-Cbl became EGFR associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment specific. In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.     

Effects of Membrane Trafficking on Signaling by Receptor Tyrosine Kinases

The intracellular trafficking machinery contributes to the spatial and temporal control of signaling by receptor tyrosine kinases (RTKs). The primary role in this process is played by endocytic trafficking, which regulates the localization of RTKs and their downstream effectors, as well as the duration and the extent of their activity. The key regulatory points along the endocytic pathway are internalization of RTKs from the plasma membrane, their sorting to degradation or recycling, and their residence in various endosomal compartments. Here I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. Cumulatively, these mechanisms illustrate a multilayered control of RTK signaling exerted by the trafficking machinery.At the cellular level, receptor tyrosine kinases (RTKs) need to be properly localized to function as signal-receiving and signal-transmitting devices (Lemmon and Schlessinger 2010). To receive signals (i.e., to bind extracellular ligands), RTKs have to be exposed at the surface of the plasma membrane. To transmit signals after ligand binding by RTKs, appropriate signaling components have to be available within intracellular compartments: in the cytoplasm, in association with membrane-bound organelles and in the cell nucleus. Importantly, the intracellular distribution of RTKs and their associated partners is not static but undergoes dynamic changes in different phases of signaling, as reflected for example by endocytic internalization of activated RTKs (Scita and Di Fiore 2010). Therefore, to function properly, the whole RTK signaling machinery within the cell has to be organized and tightly controlled both in space and in time. This organization and control are ensured by intracellular trafficking machineries, mainly by membrane transport systems such as endocytosis and secretion but also by other distribution systems (e.g., responsible for nucleocytoplasmic shuttling of proteins).Recent years have brought increasing evidence that intracellular membrane trafficking, in particular endocytic internalization, degradation, and recycling, can profoundly affect the signaling properties of RTKs (Mukherjee et al. 2006; Abella and Park 2009; Lemmon and Schlessinger 2010; Scita and Di Fiore 2010; Grecco et al. 2011; Sigismund et al. 2012). The changes in the amounts of RTKs at the cell surface can alter the cellular responses when ligands are abundant (Grecco et al. 2011). In turn, the presence of a given RTK at the plasma membrane is determined by the rates of three trafficking processes: delivery of newly synthesized molecules by the secretory pathway, their internalization (occurring for both ligand-bound and ligand-free molecules), and endocytic recycling. Although the molecular details concerning the regulation of RTK delivery to the plasma membrane are not well known, numerous studies document various mechanisms by which internalization and recycling of RTKs can be modulated, thus affecting the signaling outputs (Le Roy and Wrana 2005). In addition to the regulation of RTKs at the cell surface, trafficking processes control the intracellular fate of endocytosed RTKs. Following internalization, RTKs can be either targeted for lysosomal degradation, or recycled back to the plasma membrane (Mukherjee et al. 2006; Abella and Park 2009; Scita and Di Fiore 2010). The first route results in the termination of signaling, whereas the second allows for sustained signaling if the ligand is available. Usually degradation and recycling of a given RTK can occur simultaneously but the balance between them is crucial to determine the net signaling output. Again, the molecular mechanisms that can shift the fate of internalized RTKs between degradation and recycling, thus changing RTK signaling, have begun to emerge in recent years (Polo and Di Fiore 2006; von Zastrow and Sorkin 2007; Sorkin and von Zastrow 2009; Sigismund et al. 2012). Finally, in contrast to an early view that only RTKs present at the plasma membrane are signaling competent, it is now accepted that in many cases activated RTKs can emit signals also after internalization into intracellular compartments (Miaczynska et al. 2004b; Miaczynska and Bar-Sagi 2010; Platta and Stenmark 2011). In some cell types (e.g., in neurons), such “signaling endosomes” are crucial for signal propagation within the cell and for the final cellular response. Moreover, endosomes can serve as platforms for amplification and compartmentalization of signals emitted by RTKs (Sadowski et al. 2009; Platta and Stenmark 2011).In this article, I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. As the membrane trafficking system of a cell is highly interconnected and can be considered a global dynamic continuum, it is important to note that often one primary alteration at a given stage of RTK trafficking may affect other transport steps or compartments, thus causing generalized changes in the intracellular routing and signaling of RTKs.     

Differential Regulation of Cysteinyl Leukotriene Receptor Signaling by Protein Kinase C in Human Mast Cells

Cysteinyl leukotrienes (cys-LTs) are a group of lipid mediators that are potent bronchoconstrictors, powerful inducers of vascular leakage and potentiators of airway hyperresponsiveness. Cys-LTs play an essential role in asthma and are synthesized as well as activated in mast cells (MCs). Cys-LTs relay their effects mainly through two known GPCRs, CysLT₁R and CysLT₂R. Although protein kinase C (PKC) isoforms are implicated in the regulation of CysLT₁R function, neither the role of PKCs in cys-LT-dependent MC inflammatory signaling nor the involvement of specific isoforms in MC function are known. Here, we show that PKC inhibition augmented LTD₄ and LTE₄-induced calcium influx through CysLT₁R in MCs. In contrast, inhibition of PKCs suppressed c-fos expression as well MIP1β generation by cys-LTs. Interestingly, cys-LTs activated both PKCα and PKCε isoforms in MC. However, knockdown of PKCα augmented cys-LT mediated calcium flux, while knockdown of PKCε attenuated cys-LT induced c-fos expression and MIP1β generation. Taken together, these results demonstrate for the first time that cys-LT signaling downstream of CysLT₁R in MCs is differentially regulated by two distinct PKCs which modulate inflammatory signals that have significant pathobiologic implications in allergic reactions and asthma pathology.     

Regulation of Insulin Receptor Trafficking by Bardet Biedl Syndrome Proteins

Insulin and its receptor are critical for the regulation of metabolic functions, but the mechanisms underlying insulin receptor (IR) trafficking to the plasma membrane are not well understood. Here, we show that Bardet Biedl Syndrome (BBS) proteins are necessary for IR localization to the cell surface. We demonstrate that the IR interacts physically with BBS proteins, and reducing the expression of BBS proteins perturbs IR expression in the cell surface. We show the consequence of disrupting BBS proteins for whole body insulin action and glucose metabolism using mice lacking different BBS genes. These findings demonstrate the importance of BBS proteins in underlying IR cell surface expression. Our data identify defects in trafficking and localization of the IR as a novel mechanism accounting for the insulin resistance commonly associated with human BBS. This is supported by the reduced surface expression of the IR in fibroblasts derived from patients bearing the M390R mutation in the BBS1 gene.     

G蛋白偶联受体激酶的调控

G蛋白偶联受体激酶(G protein-coupled receptor kinase,GRK)特异地使活化的G蛋白偶联受体(G protein-coupled receptor,GPCR)发生磷酸化及脱敏化,从而终止后者介导的信号转导通路。研究表明,GRK的功能被高度调控,并具有下行调节GPCR的能力。调控GRK功能的机制包括两个层次：(1)多种途径调控激酶的亚细胞定位及活性,包括GPCR介导、G蛋白偶联、磷脂作用、Ca^2 结合蛋白调控、蛋白激酶C活化、MAPK反馈抑制、小窝蛋白抑制等;(2)调控GRK表达水平,主要体现在其与某些疾病的联系。     

Dysregulation of ErbB Receptor Trafficking and Signaling in Demyelinating Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy with the majority of cases involving demyelination of peripheral nerves. The pathogenic mechanisms of demyelinating CMT remain unclear, and no effective therapy currently exists for this disease. The discovery that mutations in different genes can cause a similar phenotype of demyelinating peripheral neuropathy raises the possibility that there may be convergent mechanisms leading to demyelinating CMT pathogenesis. Increasing evidence indicates that ErbB receptor-mediated signaling plays a major role in the control of Schwann cell-axon communication and myelination in the peripheral nervous system. Recent studies reveal that several demyelinating CMT-linked proteins are novel regulators of endocytic trafficking and/or phosphoinositide metabolism that may affect ErbB receptor signaling. Emerging data have begun to suggest that dysregulation of ErbB receptor trafficking and signaling in Schwann cells may represent a common pathogenic mechanism in multiple subtypes of demyelinating CMT. In this review, we focus on the roles of ErbB receptor trafficking and signaling in regulation of peripheral nerve myelination and discuss the emerging evidence supporting the potential involvement of altered ErbB receptor trafficking and signaling in demyelinating CMT pathogenesis and the possibility of modulating these trafficking and signaling processes for treating demyelinating peripheral neuropathy.     

Regulation of the Hippo-YAP Pathway by G-Protein-Coupled Receptor Signaling

The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations.     

Receptor Tyrosine Kinase Signaling and Primordial Germ Cell Development

Primordial germ cells (PGCs) give rise to sperms and eggs. Their development is crucial to species propagation and has to be precisely controlled. Studies in several model organisms have identified many genes involved in the specification and guided migration of PGCs. However, the mechanisms governing the behaviors of this unique type of cells remain to be investigated. Interestingly, PGCs share certain cellular properties with metastasizing cancer cells including proliferation, invasion of other tissues, survival, and migration. Recently we have shown that in Drosophila the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development. In later stages, activation of both STAT and Ras, likely by other molecules, is required continuously for PGC migration. The requirement for RTK suggests molecular conservation between flies and mice in PGC development and also suggests that germ cells and cancer cells share certain intracellular signaling strategies.     

Regulation of Receptor Tyrosine Kinase Ligand Processing

A primary mode of regulating receptor tyrosine kinase (RTK) signaling is to control access of ligand to its receptor. Many RTK ligands are synthesized as transmembrane proteins. Frequently, the active ligand must be released from the membrane by proteolysis before signaling can occur. Here, we discuss RTK ligand shedding and describe the proteases that catalyze it in flies and mammals. We focus principally on the control of EGF receptor ligand shedding, but also refer to ligands of other RTKs. Two prominent themes emerge. First, control by regulated trafficking and cellular compartmentalization of the proteases and their ligand substrates plays a key role in shedding. Second, many external signals converge on the shedding proteases and their control machinery. Proteases therefore act as regulatory hubs that integrate information that the cell receives and translate it into precise outgoing signals. The activation of signaling by proteases is therefore an essential element of the cellular communication machinery.Cells must talk to one another. This principle applies throughout the tree of life: from unicellular bacteria, to the trillions of cells that coordinate to make a mammal. Communication between cells requires dedicated machinery, capable of relaying information across membranes. Transmembrane proteins are therefore essential for signaling. Understanding how this is regulated is paramount. In mammals, receptor tyrosine kinases (RTKs) and their ligands are important examples of such machinery (Schlessinger 2000), controlling many biological processes including development, immunity, tissue repair, and metabolic homeostasis (Ullrich and Schlessinger 1990). They are transmembrane proteins with an extracellular ligand-binding motif and an intracellular kinase domain. As discussed in other chapters, a common mode of RTK activation involves receptor dimerization induced by ligand binding (Lemmon and Schlessinger 2010).Regulated access of ligand to receptor, over distance and time, is key to controlling signaling. Ligands are frequently synthesized as transmembrane forms; when they remain membrane-tethered and cannot diffuse, the range over which they can operate is limited to adjacent cells (Massague and Pandiella 1993; Singh and Harris 2005). Other ligands are soluble secretory proteins. This enables paracrine and endocrine signaling—communication between nonadjacent cells. A more complex mode of signaling exploits the characteristics of both of the above. Ligand is synthesized as a transmembrane precursor, which is then shed from the cell surface by proteolysis. This adds an additional and stringent regulatory step to a signaling network (Massague and Pandiella 1993).This chapter will focus on RTK ligand cleavage and its regulation. We shall highlight how shedding is often critical for signaling, and describe the protease families that catalyze ligand release in flies and mammals. An emergent theme is that regulated trafficking and compartmentalization of ligand and protease modulate signaling. Another theme will be the range of stimuli that impinge on shedding.The epidermal growth factor receptor (EGFR) is an excellent model RTK to illustrate the regulation of ligand proteolysis because the requirement for ligand cleavage in signaling is well established, and the major physiological sheddases have been identified (Blobel 2005). Where warranted, physiological evidence for the role of ligand shedding in the regulation of other RTKs will also be discussed. Whereas we shall deal mostly with ADAM proteases (“a disintegrin and metalloprotease”), which represent the canonical mammalian RTK ligand shedding machinery, the rhomboid family of intramembrane proteases will also be discussed.     

Tie2 and Eph Receptor Tyrosine Kinase Activation and Signaling

The Eph and Tie cell surface receptors mediate a variety of signaling events during development and in the adult organism. As other receptor tyrosine kinases, they are activated on binding of extracellular ligands and their catalytic activity is tightly regulated on multiple levels. The Eph and Tie receptors display some unique characteristics, including the requirement of ligand-induced receptor clustering for efficient signaling. Interestingly, both Ephs and Ties can mediate different, even opposite, biological effects depending on the specific ligand eliciting the response and on the cellular context. Here we discuss the structural features of these receptors, their interactions with various ligands, as well as functional implications for downstream signaling initiation. The Eph/ephrin structures are already well reviewed and we only provide a brief overview on the initial binding events. We go into more detail discussing the Tie-angiopoietin structures and recognition.     

Regulation of G Protein-Coupled Receptor Signaling by A-Kinase Anchoring Proteins

Specificity of transduction events is controlled at the molecular level by scaffold, anchoring, and adaptor proteins, which position signaling enzymes at proper subcellular localization. This allows their efficient catalytic activation and accurate substrate selection. A-kinase anchoring proteins (AKAPs) are group of functionally related proteins that compartmentalize the cAMP-dependent protein kinase (PKA) and other signaling enyzmes at precise subcellular sites in close proximity to their physiological substrate(s) and favor specific phosphorylation events. Recent evidence suggests that AKAP transduction complexes play a key role in regulating G protein-coupled receptor (GPCR) signaling. Regulation can occur at multiple levels because AKAPs have been shown both to directly modulate GPCR function and to act as downstream effectors of GPCR signaling. In this minireview, we focus on the molecular mechanisms through which AKAP-signaling complexes modulate GPCR transduction cascades.     

PI3-Kinase Is Involved in Mitogenic Signaling by the Oncogenic Receptor Tyrosine Kinase Xiphophorus Melanoma Receptor Kinase in Fish Melanoma

Overexpression of the mutationally activated receptor tyrosine kinase Xiphophorus melanoma receptor kinase (Xmrk) initiates formation of hereditary malignant melanoma in the fish Xiphophorus. In melanoma as well as in a melanoma-derived cell line (PSM) this receptor is highly activated resulting in constitutive Xmrk-mediated mitogenic signaling. In order to analyze mitogenic signaling triggered by Xmrk a possible involvement of phosphatidylinositol 3 (PI3)-kinase in Xmrk signal transduction was examined. Constitutive binding of the p85 adapter subunit of PI3-kinase to the Xmrk receptor was detected in PSM melanoma cells. Further analyses in BHK cells expressing a Xmrk chimera (HER-mrk) showed that p85 association with the intracellular part of Xmrk was dependent on autophosphorylation of the receptor. In vitro binding studies revealed that the interaction is mediated mainly through the N-terminal SH2 domain of p85 which directly binds to a sequence motif around phosphorylated Tyr-983 in the Xmrk carboxy-terminus. In accordance with recruitment of p85 by Xmrk in PSM cells, the PI3-kinase downstream target Akt was found to be highly phosphorylated on Ser-473, indicating efficient PI3-kinase signaling in melanoma cells. PI3-kinase activation was also detected in Xiphophorus melanoma. Moreover, malignant melanomas exhibited an increased level of PI3-kinase activity which was about three times higher than that in benign pigmented lesions. Inhibition of PI3-kinase activity in PSM melanoma cells by both Wortmannin and LY294002 blocked entry into S-phase. Together these data demonstrate that PI3-kinase is a substrate of the oncogenic Xmrk receptor and plays a significant role in mitogenic signaling of melanoma cells and the formation of malignant melanoma in Xiphophorus.     

Casein Kinase 2 Dependent Phosphorylation of Neprilysin Regulates Receptor Tyrosine Kinase Signaling to Akt

Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling.     

A GIPC1-Palmitate Switch Modulates Dopamine Drd3 Receptor Trafficking and Signaling

Palmitoylation is involved in several neuropsychiatric and movement disorders for which a dysfunctional signaling of the dopamine D3 receptor (Drd3) is hypothesized. Computational modeling of Drd3''s homologue, Drd2, has shed some light on the putative role of palmitoylation as a reversible switch for dopaminergic receptor signaling. Drd3 is presumed to be palmitoylated, based on sequence homology with Drd2, but the functional attributes afforded by Drd3 palmitoylation have not been studied. Since these receptors are major targets of antipsychotic and anti-Parkinsonian drugs, a better characterization of Drd3 signaling and posttranslational modifications, like palmitoylation, may improve the prospects for drug development. Using molecular dynamics simulations, we evaluated in silico how Drd3 palmitoylation could elicit significant remodeling of the C-terminal cytoplasmic domain to expose docking sites for signaling proteins. We tested this model in cellulo by using the interaction of Drd3 with the G-alpha interacting protein (GAIP) C terminus 1 (GIPC1) as a template. From a series of biochemical studies, live imaging, and analyses of mutant proteins, we propose that Drd3 palmitoylation acts as a molecular switch for Drd3-biased signaling via a GIPC1-dependent route, which is likely to affect the mode of action of antipsychotic drugs.     

Dorsal Ruffle Microdomains Potentiate Met Receptor Tyrosine Kinase Signaling and Down-regulation

Dorsal ruffles are apical protrusions induced in response to many growth factors, yet their function is poorly understood. Here we report that downstream from the hepatocyte growth factor (HGF) receptor tyrosine kinase (RTK), Met, dorsal ruffles function as both a localized signaling microdomain as well as a platform from which the Met RTK internalizes and traffics to a degradative compartment. In response to HGF, colonies of epithelial Madin-Darby canine kidney cells form dorsal ruffles for up to 20 min. Met is transcytosed from the basolateral membrane on Rab4 endosomes, to the apical surface where Met, as well as a Met substrate and scaffold protein, Gab1, localize to the dorsal ruffle membrane. This results in activation of downstream signaling proteins, as evidenced by localization of phospho-ERK1/2 to dorsal ruffles. As dorsal ruffles collapse, Met is internalized into EEA1- and Rab5-positive endosomes and is targeted for degradation through delivery to an Hrs-positive sorting compartment. Enhancing HGF-dependent dorsal ruffle formation, through overexpression of Gab1 or activated Pak1 kinase, promotes more efficient degradation of the Met RTK. Conversely, the ablation of dorsal ruffle formation, by pre-treatment with SITS (4-acetamido-4′-isothiocyabatostilbene-2′,2-disulfonic acid) or expression of a Gab1 mutant, impairs Met degradation. Taken together, these data support a function for dorsal ruffles as a biologically relevant signaling microenvironment and a mechanism for Met receptor internalization and degradation.     

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.