The neurotrophin receptor signaling endosome: Where trafficking meets signaling

Neurons are the largest cells in the body and form subcellular compartments such as axons and dendrites. During both development and adulthood building blocks must be continually trafficked long distances to maintain the different regions of the neuron. Beyond building blocks, signaling complexes are also transported, allowing for example, axons to communicate with the soma. The critical roles of signaling via ligand–receptor complexes is perhaps best illustrated in the context of development, where they are known to regulate polarization, survival, axon outgrowth, dendrite development, and synapse formation. However, knowing ‘when’ and ‘how much’ signaling is occurring does not provide the complete story. The location of signaling has a significant impact on the functional outcomes. There are therefore complex and functionally important trafficking mechanisms in place to control the precise spatial and temporal aspects of many signal transduction events. In turn, many of these signaling events affect trafficking mechanisms, setting up an intricate connection between trafficking and signaling. In this review we will use neurotrophin receptors, specifically TrkA and TrkB, to illustrate the cell biology underlying the links between trafficking and signaling. Briefly, we will discuss the concepts of how trafficking and signaling are intimately linked for functional and diverse signaling outputs, and how the same protein can play different roles for the same receptor depending on its localization. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Regulator of G protein signaling 6 (RGS6) protein ensures coordination of motor movement by modulating GABAB receptor signaling

γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABA(B) receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gβγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABA(B)R signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ(5) and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABA(B)R antagonist. RGS6(-/-) mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABA(B)R, and GIRK channel subunits, and cerebellar granule neurons from RGS6(-/-) mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABA(B)R signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.     

Illuminating the early signaling pathway of a fungal light‐oxygen‐voltage photoreceptor

Circadian clocks are molecular timekeepers encountered in a wide variety of organisms, which allow to adapt the cell's metabolism and behavior to the daily and seasonal periods. Their function is regulated by light‐sensing proteins, among which Vivid, a light‐oxygen‐voltage (LOV) sensitive domain of the fungus Neurospora crassa, constitutes one of the most prominent examples. Although the major photochemical and structural changes during the photocycle of this photosensor have been elucidated through experimental means, its signal transduction pathway is still poorly resolved at the molecular level. In this article, we show through molecular dynamics simulation that the primary steps after adduct formation involve a switch of Gln182 in vicinity of the chromophore FAD (flavin–adenine–dinucleotide), followed by a coupling between the Iβ‐ and Hβ‐strands through H‐bond formation between Gln182 and Asn161 as well as subsequent weakening of the H‐bonding interaction between the Iβ‐ and Aβ‐strands. These processes then induce a reorientation of the Aβ‐Bβ‐loop with respect to the protein core as well as a simultaneous contraction of the partially unfolded α‐helix onto the α‐Aβ‐linker at the Ncap. Finally, we demonstrate through additional dimer simulations that the light‐induced conformational changes, observed in the monomeric case, play a decisive role in controlling the dimerization tendency of Vivid with its partner domains and that the light‐state homodimer shows a much larger affinity for aggregation than the dark state. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Fibronectin inhibits osteoclastogenesis while enhancing osteoclast activity via nitric oxide and interleukin‐1β‐mediated signaling pathways

Osteoclasts are bone‐resorbing cells formed by fusion of mononuclear precursors. The matrix proteins, fibronectin (FN), vitronectin (VN), and osteopontin (OPN) are implicated in joint destruction and interact with osteoclasts mainly through integrins. To assess the effects of these matrix proteins on osteoclast formation and activity, we used RAW 264.7 (RAW) cells and mouse splenocytes differentiated into osteoclasts on tissue culture polystyrene (TCP) or osteologic? slides pre‐coated with 0.01–20 µg/ml FN, VN, and OPN. At 96 h, osteoclast number and multinucleation were decreased on VN and FN compared to OPN and TCP in both RAW and splenocytes cell cultures. When early differentiation was assessed, VN but not FN decreased cytoplasmic tartrate‐resistant acid phosphatase activity and pre‐osteoclast number at 48 h. OPN had the opposite effect to FN on osteoclast formation. When RAW cells were differentiated on OPN and treated by FN and OPN, osteoclast number only in the FN treated group was 40–60% lower than the control, while the total number of nuclei was unchanged, suggesting that FN delays osteoclast fusion. In contrast to its inhibitory effect on osteoclastogenesis, FN increased resorption by increasing both osteoclast activity and the percentage of resorbing osteoclasts. This was accompanied by an increase in nitric oxide (NO) levels and interleukin‐1β (IL‐1β). IL‐1β production was inhibited using the NO‐synthase inhibitor only on FN indicating a FN‐specific cross‐talk between NO and IL‐1β signaling pathways. We conclude that FN upregulates osteoclast activity despite inhibiting osteoclast formation and that these effects involve NO and IL‐1β signaling. J. Cell. Biochem. 111: 1020–1034, 2010. © 2010 Wiley‐Liss, Inc.     

Insulin signaling in microdomains of the plasma membrane

Although the effects of insulin on glucose and lipid metabolism are well documented, gaps remain in our understanding of the precise molecular mechanisms of signal transduction. Recent evidence suggests that compartmentalization of signaling molecules and metabolic enzymes may explain the unique cellular effects of the hormone. Signal initiation from the insulin receptor is restricted in part to caveolae microdomains of the plasma membrane. A fraction of the insulin receptor directly interacts with caveolin, thus directing the protein to caveolae. Following its activation by insulin, the receptor recruits a series of adapter proteins, resulting in the activation of the G protein TC10, which also resides in caveolae. TC10 can influence a number of cellular processes, including changes in the actin cytoskeleton, recruitment of effector including the adapter protein CIP4, and assembly of the exocyst complex. These events play crucial roles in the trafficking, docking and fusion of vesicles containing the insulin-responsive glucose transporter Glut4 at the plasma membrane.     

Intracellular regulation of heterotrimeric G-protein signaling modulates vascular smooth muscle cell contraction

The contractile function of vascular smooth muscle cells within the media of resistance arterioles is tightly connected to the role of these blood vessels in the maintenance of blood pressure homeostasis. Thus, much effort has been made to understand the intracellular signaling pathways that control vascular smooth muscle cell contractility with the aim that this knowledge will provide important clues for reducing the impact of uncontrolled blood pressure in our society. A key set of surface receptors, the G-protein coupled receptors, has been widely associated with the regulation of vascular smooth muscle cell contractility. Indeed, many of the current treatments for hypertension involve selective inhibition of these receptors. More recently, we have begun to understand the cellular mechanisms whereby G-protein coupled pathways are connected to the contractile machinery of the vascular smooth muscle cells. What has emerged is a view where there are multiple intracellular control points for G-protein signaling that coordinate and focus the extracellular stimuli into meaningful physiologic responses. This work will examine some of the recent advances in our understanding of G-protein signaling and its regulation of contractile function in vascular smooth muscle cells.     

Microbe‐inducible trafficking pathways that control Toll‐like receptor signaling

The receptors of the mammalian innate immune system are designed for rapid microbial detection, and are located in organelles that are conducive to serve these needs. However, emerging evidence indicates that the sites of microbial detection are not the sites of innate immune signal transduction. Rather, microbial detection triggers the movement of receptors to regions of the cell where factors called sorting adaptors detect active receptors and promote downstream inflammatory responses. These findings highlight the critical role that membrane trafficking pathways play in the initiation of innate immunity to infection. In this review, we describe pathways that promote the microbe‐inducible endocytosis of Toll‐like receptors (TLRs), and the microbe‐inducible movement of TLRs between intracellular compartments. We highlight a new class of proteins called Transporters Associated with the eXecution of Inflammation (TAXI), which have the unique ability to transport TLRs and their microbial ligands to signaling‐competent regions of the cell, and we discuss the means by which the subcellular sites of signal transduction are defined.      

EVL regulates VEGF receptor‐2 internalization and signaling in developmental angiogenesis

Endothelial tip cells are essential for VEGF‐induced angiogenesis, but underlying mechanisms are elusive. The Ena/VASP protein family, consisting of EVL, VASP, and Mena, plays a pivotal role in axon guidance. Given that axonal growth cones and endothelial tip cells share many common features, from the morphological to the molecular level, we investigated the role of Ena/VASP proteins in angiogenesis. EVL and VASP, but not Mena, are expressed in endothelial cells of the postnatal mouse retina. Global deletion of EVL (but not VASP) compromises the radial sprouting of the vascular plexus in mice. Similarly, endothelial‐specific EVL deletion compromises the radial sprouting of the vascular plexus and reduces the endothelial tip cell density and filopodia formation. Gene sets involved in blood vessel development and angiogenesis are down‐regulated in EVL‐deficient P5‐retinal endothelial cells. Consistently, EVL deletion impairs VEGF‐induced endothelial cell proliferation and sprouting, and reduces the internalization and phosphorylation of VEGF receptor 2 and its downstream signaling via the MAPK/ERK pathway. Together, we show that endothelial EVL regulates sprouting angiogenesis via VEGF receptor‐2 internalization and signaling.     

Elements of the olfactory signaling pathways in insect antennae

Owing to their enormous ability to recognize airborne molecules, insects have long been used as model systems for studying various aspects of olfaction. Modern biological techniques have opened new avenues for exploring the molecular mechanisms underlying the complex signaling processes in chemosensory neurons. Biochemical and molecular analyses have allowed the identification of molecular elements of the olfactory reaction pathways and have shed light on mechanisms that account for the sensitivity and specificity of the chemosensory system.     

bFGF and LIF signaling activates STAT3 in proliferating myoblasts

Different mitogens elicit similar effects on growth and differentiation of skeletal muscle, suggesting that potential overlap exists in the signaling cascades activated by such factors. To investigate this possibility, we examined the status of STAT and ERK proteins in C2C12 myoblasts and myotubes following stimulation with bFGF or LIF. Both STAT1 and STAT3 as well as ERK1 and ERK2 proteins were detectable in extracts of myoblasts. LIF stimulation of myoblasts lead to rapid phosphorylation on tyrosine of STAT3 and of ERKs 1 and 2. Similarly, bFGF stimulation of myoblasts resulted in the tyrosine phosphorylation of STAT3. However, unlike LIF, the bFGF induced tyrosine phosphorylation of STAT3 appeared cyclical, with recurrent peaks of phosphorylation even after prolonged exposure. By contrast, STAT1 remained unphosphorylated in myoblasts treated with bFGF or LIF. In differentiated myotubes, LIF treatment resulted in the tyrosine phosphorylation of both STAT3 and STAT1, but ERK phosphorylation was not detectable, and bFGF treatment did not lead to STAT1 or STAT3 tyrosine phosphorylation. Therefore these observations suggest that disparate mitogens can activate similar downstream effectors in proliferating myoblasts. © 1996 Wiley-Liss, Inc.     

Non-canonical signaling and localizations of heterotrimeric G proteins

Heterotrimeric G proteins typically transduce signals from G protein-coupled receptors (GPCRs) to effector proteins. In the conventional G protein signaling paradigm, the G protein is located at the cytoplasmic surface of the plasma membrane, where, after activation by an agonist-bound GPCR, the GTP-bound Gα and free Gβγ bind to and regulate a number of well-studied effectors, including adenylyl cyclase, phospholipase Cβ, RhoGEFs and ion channels. However, research over the past decade or more has established that G proteins serve non-canonical roles in the cell, whereby they regulate novel effectors, undergo activation independently of a GPCR, and/or function at subcellular locations other than the plasma membrane. This review will highlight some of these non-canonical aspects of G protein signaling, focusing on direct interactions of G protein subunits with cytoskeletal and cell adhesion proteins, the role of G proteins in cell division, and G protein signaling at diverse organelles.     

Bacterial cupredoxin azurin hijacks cellular signaling networks: Protein–protein interactions and cancer therapy

Azurin secreted by Pseudomonas aeruginosa is an anticancer bacteriocin, which preferentially enters human cancer cells and induces apoptosis or growth inhibition. It turns out that azurin is a multi‐target anticancer agent interfering in the p53 signaling pathway and the non‐receptor tyrosine kinases signaling pathway. This suggests that azurin exerts its anticancer activity by interacting with multiple targets and interfering in multiple steps in disease progression. Therefore, azurin could overcome resistance to therapy. Besides azurin, putative bacteriocins that possess functional properties similar to those of azurin have been identified in more bacteria species. A systematic investigation on the anticancer mechanisms of azurin and the azurin‐like bacteriocins will provide more and better options in cancer therapy. In this review, we summarize how azurin and the derived peptides hijack key cellular regulators or cell surface receptors to remodel the cellular signaling networks. In particular, we highlight the necessity of determining the structure of azurin/p53 complex and investigating the influence of post‐translational modifications on interactions between azurin and p53. Therapeutic applications of azurin and derived peptides are also discussed.     

Interrogating cyclic AMP signaling using optical approaches

Optical reporters for cAMP represent a fundamental advancement in our ability to investigate the dynamics of cAMP signaling. These fluorescent sensors can measure changes in cAMP in single cells or in microdomains within cells as opposed to whole populations of cells required for other methods of measuring cAMP. The first optical cAMP reporters were FRET-based sensors utilizing dissociation of purified regulatory and catalytic subunits of PKA, introduced by Roger Tsien in the early 1990s. The utility of these sensors was vastly improved by creating genetically encoded versions that could be introduced into cells with transfection, the first of which was published in the year 2000. Subsequently, improved sensors have been developed using different cAMP binding platforms, optimized fluorescent proteins, and targeting motifs that localize to specific microdomains. The most common sensors in use today are FRET-based sensors designed around an Epac backbone. These rely on the significant conformational changes in Epac when it binds cAMP, altering the signal between FRET pairs flanking Epac. Several other strategies for optically interrogating cAMP have been developed, including fluorescent translocation reporters, dimerization-dependent FP based biosensors, BRET (bioluminescence resonance energy transfer)-based sensors, non-FRET single wavelength reporters, and sensors based on bacterial cAMP-binding domains. Other newly described mammalian cAMP-binding proteins such as Popdc and CRIS may someday be exploited in sensor design. With the proliferation of engineered fluorescent proteins and the abundance of cAMP binding targets in nature, the field of optical reporters for cAMP should continue to see rapid refinement in the coming years.     

Evolutionarily conserved DELLA-mediated gibberellin signaling in plants

Gibberellins (GAs) play important roles in many essential plant growth and development processes. A family of nuclear growth-repressing DELLA proteins is the key component in GA signaling. GA perception is mediated by GID1, and the key event of GA signaling is the degradation of DELLA proteins via the 26S proteasome pathway. DELLA proteins integrating other plant hormones signaling and environmental cue modulating plant growth and development have been revealed. GA turning on the de-DELLA-repressing system is conserved, and independently establishes step-by-step recruitment of GA-stimulated GID1-DELLA interaction and DELLA growth-repression functions during land plant evolution. These discoveries open new prospects for the understanding of GA action and DELLA-mediated signaling in plants.     

ERM proteins: from cellular architecture to cell signaling

ERM (ezrin/radixin/moesin) proteins, concentrated in actin rich cell-surface structures, cross-link actin filaments with the plasma membrane. They are involved in the formation of microvilli, cell-cell adhesion, maintenance of cell shape, cell motility and membrane trafficking. Recent analyses reveal that they are not only involved in cytoskeleton organization but also in signaling pathway. They play an important role in the activation of members of the Rho family by recruiting their regulators. The functions of ERM proteins are regulated by their conformational charges: the intramolecular interaction between the N- and C-terminal domains of ERM proteins charges masks several binding sites, leading to a dormant protein. Different activation signals regulate ERM proteins functions by modulating these intramolecular interactions. The involvement of ERM proteins in many signaling pathways has led to study their role during development of different species.     

BMP signaling in vascular biology and dysfunction

The vascular system is critical for developmental growth, tissue homeostasis and repair but also for tumor development. Bone morphogenetic protein (BMP) signaling has recently emerged as a fundamental pathway of the endothelium by regulating cardiovascular and lymphatic development and by being causative for several vascular dysfunctions. Two vascular disorders have been directly linked to impaired BMP signaling: pulmonary arterial hypertension and hereditary hemorrhagic telangiectasia. Endothelial BMP signaling critically depends on the cellular context, which includes among others vascular heterogeneity, exposure to flow, and the intertwining with other signaling cascades (Notch, WNT, Hippo and hypoxia). The purpose of this review is to highlight the most recent findings illustrating the clear need for reconsidering the role of BMPs in vascular biology.     

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.