Regulation of receptor tyrosine kinase signaling by endocytic trafficking

Activated receptor tyrosine kinase (RTK) receptors are rapidly internalized and eventually delivered to the lysosomes. Although ligand-induced endocytosis was originally thought to be a mechanism of receptor inactivation, many studies suggest that receptors remain active within endosomes. This review discusses the role that internalized signaling complexes may play in different RTK systems including recent data on how ubiquitination may regulate this process. In general, it appears that some receptor systems have evolved to enhance endosomal signaling, as is the case for TrkA and NGF. In contrast, the insulin receptor system appears to limit the extent of endosomal signaling. The EGFR system is the intermediate example. In this case, some signals are specifically generated from the cell surface while others appear to be generated from within endosomes. This may act as a mechanism to produce ligand-specific signals. Thus, trafficking could play diverse roles in receptor signaling, depending on the specific cell and tissue type.     

NMDA受体信号复合体中蛋白质的相互作用

谷氨酸能兴奋性突触的突触后密集区(postsynaptic density,PSD)包含多种受体蛋白、骨架蛋白和信号蛋白,它们通过分子中特定的结构域相互识别并动态地结合,形成多个信号复合体,参与突触后受体功能的调节及其下游特异性信号转导通路的激活。其中,NMDA受体信号复合体中蛋白质-蛋白质的相互作用及其调控机制的阐明,对于深入了解神经发育、突触可塑性、兴奋性毒性等生理病理的分子机制有重要意义。     

VEGF signaling inside vascular endothelial cells and beyond

Vascular endothelial growth factor-A (VEGF-A) has long been recognized as the key regulator of vascular development and function in health and disease. VEGF is a secreted polypeptide that binds to transmembrane tyrosine kinase VEGF receptors on the plasma membrane, inducing their dimerization, activation and assembly of a membrane-proximal signaling complex. Recent studies have revealed that many key events of VEGFR signaling occur inside the endothelial cell and are regulated by endosomal receptor trafficking. Plasma membrane VEGFR interacting molecules, including vascular guidance receptors Neuropilins and Ephrins also regulate VEGFR endocytosis and trafficking. VEGF signaling is increasingly recognized for its roles outside of the vascular system, notably during neural development, and blood vessels regulate epithelial branching morphogenesis. We review here recent advances in our understanding of VEGF signaling and its biological roles.     

Retrolinkin cooperates with endophilin A1 to mediate BDNF-TrkB early endocytic trafficking and signaling from early endosomes

Brain-derived neurotrophic factor (BDNF) binds to its cell surface receptor TrkB to regulate differentiation, development, synaptic plasticity, and functional maintenance of neuronal cells. Binding of BDNF triggers TrkB dimerization and autophosphorylation, which provides docking sites for adaptor proteins to recruit and activate downstream signaling molecules. The molecular mechanisms underlying BDNF-TrkB endocytic trafficking crucial for spatiotemporal control of signaling pathways remain to be elucidated. Here we show that retrolinkin, a transmembrane protein, interacts with endophilin A1 and mediates BDNF-activated TrkB (pTrk) trafficking and signaling in CNS neurons. We find that activated TrkB colocalizes and interacts with the early endosome marker APPL1. Both retrolinkin and endophilin A1 are required for BDNF-induced dendrite development and acute extracellular signal-regulated kinase activation from early endosomes. Suppression of retrolinkin expression not only blocks BDNF-triggered TrkB internalization, but also prevents recruitment of endophilin A1 to pTrk vesicles trafficking through APPL1-positive endosomes. These findings reveal a novel mechanism for BDNF-TrkB to regulate signaling both in time and space through a specific membrane trafficking pathway.     

Axon guidance: receptor complexes and signaling mechanisms

The generation of a functional neuronal network requires that axons navigate precisely to their appropriate targets. Molecules that specify guidance decisions have been identified, and the signaling events that occur downstream of guidance receptors are beginning to be understood. New research shows that guidance receptor signaling can be hierarchical -- one receptor silencing the other -- thereby allowing navigating growth cones to interpret opposing guidance cues. Among the known intracellular signaling molecules shared by all guidance receptor families, Rho GTPases appear to be primary regulators of actin dynamics and growth cone guidance. Novel effector molecules complete the picture and suggest additional signaling mechanisms.     

Ubiquitin-dependent regulation of G protein-coupled receptor trafficking and signaling

G protein-coupled receptors (GPCRs) belong to one of the largest family of signaling receptors in the mammalian genome [1]. GPCRs elicit cellular responses to multiple diverse stimuli and play essential roles in human health and disease. GPCRs have important clinical implications in various diseases and are the targets of approximately 25–50% of all marketed drugs [2], [3]. Understanding how GPCRs are regulated is essential to delineating their role in normal physiology and in the pathophysiology of several diseases. Given the vast number and diversity of GPCRs, it is likely that multiple mechanisms exist to regulate GPCR function. While GPCR signaling is typically regulated by desensitization and endocytosis mediated by phosphorylation and β-arrestins, it can also be modulated by ubiquitination. Ubiquitination is emerging an important regulatory process that may have unique roles in governing GPCR trafficking and signaling. Recent studies have revealed a mechanistic link between GPCR phosphorylation, β-arrestins and ubiquitination that may be applicable to some GPCRs but not others. While the function of ubiquitination is generally thought to promote receptor endocytosis and endosomal sorting, recent studies have revealed that ubiquitination also plays an important role in positive regulation of GPCR signaling. Here, we will review recent developments in our understanding of how ubiquitin regulates GPCR endocytic trafficking and how it contributes to signal transduction induced by GPCR activation.     

Seven transmembrane receptor core signaling complexes are assembled prior to plasma membrane trafficking

Much is known about beta2-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the beta2-adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-G betagamma complexes initially associate in the endoplasmic reticulum, whereas G alpha subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes.     

Disabled‐2: A modular scaffold protein with multifaceted functions in signaling

Disabled‐2 (Dab2) is a multimodular scaffold protein with signaling roles in the domains of cell growth, trafficking, differentiation, and homeostasis. Emerging evidences place Dab2 as a novel modulator of cell–cell interaction; however, its mode of action has remained largely elusive. In this review, we highlight the relevance of Dab2 function in cell signaling and development and provide the most recent and comprehensive analysis of Dab2's action as a mediator of homotypical and heterotypical interactions. Accordingly, Dab‐2 controls the extent of platelet aggregation through various motifs within its N‐terminus. Dab2 interacts with the cytosolic tail of the integrin receptor blocking inside‐out signaling, whereas extracellular Dab2 competes with fibrinogen for integrin α_IIbβ₃ receptor binding and, thus, modulates outside‐in signaling. An additional level of regulation results from Dab2's association with cell surface lipids, an event that defines the extent of cell–cell interactions. As a multifaceted regulator, Dab2 acts as a mediator of endocytosis through its association with the [FY]xNPx[YF] motifs of internalized cell surface receptors, phosphoinositides, and clathrin. Other emerging roles of Dab2 include its participation in developmental mechanisms required for tissue formation and in modulation of immune responses. This review highlights the various novel mechanisms by which Dab2 mediates an array of signaling events with vast physiological consequences.     

Endocytosis and endosomes at the crossroads of regulating trafficking of axon outgrowth-modifying receptors

In neurons, many receptors must be localized correctly to axons or dendrites for proper function. During development, receptors for nerve growth and guidance are targeted to axons and localized to growth cones where receptor activation by ligands results in promotion or inhibition of axon growth. Signaling outcomes downstream of ligand binding are determined by the location, levels and residence times of receptors on the neuronal plasma membrane. Therefore, the mechanisms controlling the trafficking of these receptors are crucial to the proper wiring of circuits. Membrane proteins accumulate on the axonal surface by multiple routes, including polarized sorting in the trans Golgi network, sorting in endosomes and removal by endocytosis. Endosomes also play important roles in the signaling pathways for both growth-promoting and -inhibiting molecules: signaling endosomes derived from endocytosis are important for signaling from growth cones to cell bodies. Growth-promoting neurotrophins and growth-inhibiting Nogo-A can use EHD4/Pincher-dependent endocytosis at the growth cone for their respective retrograde signaling. In addition to retrograde transport of endosomes, anterograde transport to axons in endosomes also occurs for several receptors, including the axon outgrowth-promoting cell adhesion molecule L1/NgCAM and TrkA. L1/NgCAM also depends on EHD4/Pincher-dependent endocytosis for its axonal polarization. In this review, we will focus on receptors whose trafficking has been reported to be modulated by the EHD4/Pincher family of endosomal regulators, namely L1/NgCAM, Trk and Nogo-A. We will first summarize the pathways underlying the axonal transport of these proteins and then discuss the potential roles of EHD4/Pincher in mediating their endocytosis.     

An allosteric model for transmembrane signaling in bacterial chemotaxis

Bacteria are able to sense chemical gradients over a wide range of concentrations. However, calculations based on the known number of receptors do not predict such a range unless receptors interact with one another in a cooperative manner. A number of recent experiments support the notion that this remarkable sensitivity in chemotaxis is mediated by localized interactions or crosstalk between neighboring receptors. A number of simple, elegant models have proposed mechanisms for signal integration within receptor clusters. What is a lacking is a model, based on known molecular mechanisms and our accumulated knowledge of chemotaxis, that integrates data from multiple, heterogeneous sources. To address this question, we propose an allosteric mechanism for transmembrane signaling in bacterial chemotaxis based on the "trimer of dimers" model, where three receptor dimers form a stable complex with CheW and CheA. The mechanism is used to integrate a diverse set of experimental data in a consistent framework. The main predictions are: (1) trimers of receptor dimers form the building blocks for the signaling complexes; (2) receptor methylation increases the stability of the active state and retards the inhibition arising from ligand-bound receptors within the signaling complex; (3) trimer of dimer receptor complexes aggregate into clusters through their mutual interactions with CheA and CheW; (4) cooperativity arises from neighboring interaction within these clusters; and (5) cluster size is determined by the concentration of receptors, CheA, and CheW. The model is able to explain a number of seemingly contradictory experiments in a consistent manner and, in the process, explain how bacteria are able to sense chemical gradients over a wide range of concentrations by demonstrating how signals are integrated within the signaling complex.     

A neurotrophin signaling cascade coordinates sympathetic neuron development through differential control of TrkA trafficking and retrograde signaling

A fundamental question in developmental biology is how a limited number of growth factors and their cognate receptors coordinate the formation of tissues and organs endowed with enormous morphological complexity. We report that the related neurotrophins NGF and NT-3, acting through a common receptor, TrkA, are required for sequential stages of sympathetic axon growth and, thus, innervation of target fields. Yet, while NGF supports TrkA internalization and retrograde signaling from distal axons to cell bodies to promote neuronal survival, NT-3 cannot. Interestingly, final target-derived NGF promotes expression of the p75 neurotrophin receptor, in turn causing a reduction in the sensitivity of axons to intermediate target-derived NT-3. We propose that a hierarchical neurotrophin signaling cascade coordinates sequential stages of sympathetic axon growth, innervation of targets, and survival in a manner dependent on the differential control of TrkA internalization, trafficking, and retrograde axonal signaling.     

TGF-β receptors: In and beyond TGF-β signaling

Transforming growth factor β (TGF-β) plays an important role in normal development and homeostasis. Dysregulation of TGF-β responsiveness and its downstream signaling pathways contribute to many diseases, including cancer initiation, progression, and metastasis. TGF-β ligands bind to three isoforms of the TGF-β receptor (TGFBR) with different affinities. TGFBR1 and 2 are both serine/threonine and tyrosine kinases, but TGFBR3 does not have any kinase activity. They are necessary for activating canonical or noncanonical signaling pathways, as well as for regulating the activation of other signaling pathways. Another prominent feature of TGF-β signaling is its context-dependent effects, temporally and spatially. The diverse effects and context dependency are either achieved by fine-tuning the downstream components or by regulating the expressions and activities of the ligands or receptors. Focusing on the receptors in events in and beyond TGF-β signaling, we review the membrane trafficking of TGFBRs, the kinase activity of TGFBR1 and 2, the direct interactions between TGFBR2 and other receptors, and the novel roles of TGFBR3.     

Mechanisms of selective G protein–coupled receptor localization and trafficking

The trafficking of G protein–coupled receptors (GPCRs) to different membrane compartments has recently emerged as being a critical determinant of the signaling profiles of activation. GPCRs, which share many structural and functional similarities, also share many mechanisms that traffic them between compartments. This sharing raises the question of how the trafficking of individual GPCRs is selectively regulated. Here, we will discuss recent studies addressing the mechanisms that contribute to selectivity in endocytic and biosynthetic trafficking of GPCRs.     

Endocytosis of G Protein-Coupled Receptors and Their Ligands: Is There a Role in Metal Trafficking?

The prevalence of metal dysregulation in many neurodegenerative and neurocognitive disorders has compelled many studying such diseases to investigate the mechanisms underlying metal regulation in the central nervous system. Metal homoeostasis is often complex, with sophisticated, multilayered pathways in operation. G protein-coupled receptors are omnipresent on cell membranes and have intriguing mechanisms of endocytosis and trafficking that may be useful in metal homoeostasis. Indeed, many receptors and/or their cognate ligands are able to bind metals, and in many cases metals are considered to have neuromodulatory roles as a result of receptor binding. In this mini-review, we outline the structural and functional aspects of G protein-coupled receptors with a focus on the mechanisms leading to endocytosis and cellular trafficking. We further highlight how this may help in the trafficking of metal ions, notably copper.     

Membranes,trafficking, and signaling during animal development

Molecular genetics has been key in allowing developmental biologists to uncover many of the molecules that participate in pattern formation. Cell biology is now beginning to help developmental biologists in their quest to understand how these molecules interact within cells to direct tissue behavior. This is particularly true in the areas of membrane trafficking and cell motility. Recent work has shown that various trafficking events such as secretion, endocytosis, segregation in membrane microdomains, intracellular transport, and targeting to lysosomes regulate various signaling pathways. It is likely that within the context of an embryo, these trafficking events are integrated such that secreted factors reliably orchestrate many developmental decisions.     

Mnemons and the memorization of past signaling events

Current advances are raising our awareness of the diverse roles that protein condensation plays in the biology of cells. Particularly, findings in organisms as diverse as yeast and Drosophila suggest that cells may utilize protein condensation to establish long-lasting changes in cellular activities and thereby encode a memory of past signaling events. Proteins that oligomerize to confer such cellular memory have been termed ‘mnemons’. In the forming of super-assemblies, mnemons change their function and modulate the influence that the affected protein originally had on cellular processes. Because mnemon assemblies are self-templating, they allow cells to retain the memory of past decisions over larger timescales. Here, we review the mechanisms behind the formation of cellular memory with an emphasis on mnemon-mediated memorization of past signaling events.     

The mechanochemistry of integrated motor protein complexes

The assembly of molecular motor proteins into multi-unit protein complexes plays an important role in determining the intracellular transport and trafficking properties of many subcellular commodities. Yet, it is not known how proteins within these complexes interact and function collectively. Considering the established ties between motor transport and diseases, it has become increasingly important to investigate the functional properties of these essential transport ‘motifs’. Doing so requires that the composite motile and force-generating properties of multi-unit motor assemblies are characterized. However, such analyses are typically confounded by a lack of understanding of the links between the structural and mechanical properties of many motor complexes. New experimental challenges also emerge when one examines motor cooperation. Distributions in the mechanical microstates available to motor ensembles must be examined in order to fully understand the transport behavior of multi-motor complexes. Furthermore, mechanisms by which motors communicate must be explored to determine whether motor groups can move cargo together in a truly cooperative fashion. Resolving these issues requires the development of experimental methods that allow the dynamics of complex systems of transport proteins to be monitored with the same precision available to single-molecule biophysical assays. Herein, we discuss key fundamental principles governing the function of motor complexes and their relation to mechanisms that regulate intracellular cargo transport. We also outline new experimental strategies to resolve these essential features of intracellular transport.