Contacts between membrane proximal regions of the PDGF receptor ectodomain are required for receptor activation but not for receptor dimerization

The mechanism of PDGF-receptor beta (PDGFRbeta) activation was explored by analyzing the properties of mutant receptors designed based on the crystal structure of the extracellular region of the related receptor tyrosine kinase KIT/stem cell factor receptor. Here, we demonstrate that PDGF-induced activation of a PDGFRbeta mutated in Arg-385 or Glu-390 in D4 (the fourth Ig-like domain of the extracellular region) was compromised, resulting in impairment of a variety of PDGF-induced cellular responses. These experiments demonstrate that homotypic D4 interactions probably mediated by salt bridges between Arg-385 and Glu-390 play an important role in activation of PDGFRbeta and all type III receptor tyrosine kinases. We also used a chemical cross-linking agent to covalently cross-link PDGF-stimulated cells to demonstrate that a Glu390Ala mutant of PDGFRbeta undergoes typical PDGF-induced receptor dimerization. However, unlike WT PDGFR that is expressed on the surface of ligand-stimulated cells in an active state, PDGF-induced Glu390Ala dimers are inactive. Although the conserved amino acids that are required for mediating D4 homotypic interactions are crucial for PDGFRbeta activation, these interactions are dispensable for PDGFRbeta dimerization. Moreover, PDGFRbeta dimerization is necessary but not sufficient for tyrosine kinase activation.     

The GAS6-AXL signaling pathway triggers actin remodeling that drives membrane ruffling,macropinocytosis, and cancer-cell invasion

AXL, a member of the TAM (TYRO3, AXL, MER) receptor tyrosine kinase family, and its ligand, GAS6, are implicated in oncogenesis and metastasis of many cancer types. However, the exact cellular processes activated by GAS6-AXL remain largely unexplored. Here, we identified an interactome of AXL and revealed its associations with proteins regulating actin dynamics. Consistently, GAS6-mediated AXL activation triggered actin remodeling manifested by peripheral membrane ruffling and circular dorsal ruffles (CDRs). This further promoted macropinocytosis that mediated the internalization of GAS6-AXL complexes and sustained survival of glioblastoma cells grown under glutamine-deprived conditions. GAS6-induced CDRs contributed to focal adhesion turnover, cell spreading, and elongation. Consequently, AXL activation by GAS6 drove invasion of cancer cells in a spheroid model. All these processes required the kinase activity of AXL, but not TYRO3, and downstream activation of PI3K and RAC1. We propose that GAS6-AXL signaling induces multiple actin-driven cytoskeletal rearrangements that contribute to cancer-cell invasion.

Metastasis, the ability of cancer cells to spread from the primary tumor and invade distant secondary sites, makes cancer incurable. Despite much progress in oncology in the last decades, metastasis still causes ∼90% of cancer-related deaths. To initiate metastasis, cancer cells need first to disassemble cell–cell and cell–substrate adhesion sites and prepare for migration and invasion through the extracellular matrix (ECM), vessels, and tissues. This requires, among other things, a significant remodeling of the plasma membrane and actin cytoskeleton (1, 2).During migration and invasion, cancer cells form various actin-based protrusions such as lamellipodia, filopodia, invadopodia, peripheral ruffles (PRs), and circular dorsal ruffles (CDRs) (3–5). CDRs are enigmatic actin-rich, ring-shaped structures formed transiently on the dorsal surface of cells in response to certain growth factors. To date, it was demonstrated that CDRs are formed upon stimulation with platelet-derived growth factor (PDGF) in fibroblasts, hepatocyte growth factor (HGF) in HeLa and polarized epithelial Madin-Darby Canine Kidney (MDCK) cells, and epidermal growth factor (EGF) in fibroblasts and liver-derived epithelial cells (6–10). The functions of these structures are still not fully explored, but they have been postulated to play a role in the preparation of cells for motility, mesenchymal migration through ECM, and macropinocytosis (9). Additionally, CDRs have recently been proposed to amplify AKT activation (11).Macropinocytosis is an evolutionarily conserved, actin-dependent form of endocytosis that mediates nonselective uptake of a large amount of extracellular fluid into cells. During macropinocytosis, PRs collapse inward to create large plasma membrane–derived vesicles, termed macropinosomes, which contain extracellular fluid and solutes (12, 13). Macropinosomes may also form concomitantly with the contraction and closure of CDRs (4). Generally, macropinocytosis allows rapid and efficient remodeling of the plasma membrane and its composition. Another proposed function of macropinocytosis is to support cellular metabolism, particularly of cancer cells in which macropinocytosis is constitutively activated by mutated RAS, PTEN, or PI3K (14–17). Up-regulated macropinocytosis enables cancer cells to acquire extracellular macromolecules which, upon lysosomal degradation, provide nutrients for the metabolism and cell growth. In this way, macropinocytosis allows cancer cells to survive in a nutrient-poor tumor microenvironment (14–20). Moreover, growth factor–induced macropinocytosis has been shown to increase cell growth and proliferation by the delivery of amino acids into endolysosomes that subsequently activate mTORC1 (21).AXL is a receptor tyrosine kinase (RTK) implicated in oncogenesis. Together with TYRO3 and MER, it belongs to the TAM family. TAM receptors are dispensable for embryonic development but participate in phagocytic clearance of apoptotic cells (efferocytosis) in adult organisms (22–25). Two known TAM ligands are vitamin K–dependent proteins: growth arrest–specific 6 (GAS6) and anticoagulant protein S (PROS1). GAS6 appears to bind all three TAMs, with the highest affinity for AXL, whereas PROS1 predominantly binds TYRO3 and MER (26, 27).AXL is associated with the pathogenesis of a wide array of human cancers including gliomas, melanomas, breast, lung, and ovarian cancer (28–31). Overexpression of AXL and/or GAS6 has been shown to correlate with a poorer prognosis and increased cancer invasiveness—for example, in glioblastoma patients (32). Inhibition of AXL reduced glioma-cell migration, invasion, and proliferation in vitro and prolonged the survival of mice after intracerebral implantation of glioma cells (33, 34). An increased expression of AXL in highly metastatic breast cancer was found to be essential for all steps of the metastatic process, starting with intravasation of cancer cells (35, 36). Consistent with the association of AXL with cancer invasion and metastasis, a recent study by Revach et al. (37) reported that AXL may regulate the formation of invadopodia in melanoma cells. Furthermore, AXL has been linked to epithelial-to-mesenchymal transition (EMT) that is associated with metastasis (36, 38, 39). Several studies showed that AXL activation associated with an EMT-like phenotype conferred resistance to both conventional and targeted anticancer therapies (28, 29). Thus, AXL inhibition constitutes a promising therapeutic strategy (28, 40). Accordingly, R428, a first-in-class AXL kinase inhibitor, is being tested in the second phase of clinical trials for metastatic lung and triple-negative breast cancer, glioblastoma, and acute myeloid leukemia (41, 42).Despite the multiple roles of AXL in cancer invasion and metastasis, the molecular mechanisms underlying its action in cancer cells are not fully characterized. Here, we identified an interactome of AXL using a proximity-dependent biotin identification (BioID) assay. Our results reveal intracellular processes induced by GAS6-AXL signaling and mechanisms underlying GAS6-AXL–driven cell invasion.     

Models of dissociable receptors applicable to cyclic AMP-dependent protein kinases and membrane receptors.

We present the theory of ligand binding to protein aggregates, consisting of two types of subunits, one with a binding site for the ligand which when occupied leads to the dissociation of the other subunit from the aggregate. This theory applies to a variety of enzymes, such as protein kinases, which exist in an inhibited state because they are associated with a regulatory unit which exerts a negative constraint: binding of a regulatory ligand to this unit can dissociate the complex and release the free active form of the enzyme.These dissociable systems can exhibit remarkable properties, some of them depending on the precise stoichiometry of the binding process: (1) The concentration of free ligand for which half the binding sites are occupied increases as a function of the total concentration of protein aggregate. (2) The fact that ligand promotes the dissociation of the aggregate generates negative cooperativity in binding, so that generally the saturation curves extend over more than two orders of magnitude; in some particular cases they can exhibit an intermediary plateau. (3) Some particular stoichiometric schemes provide a nonlinear relationship between binding and effect. (4) The dissociation of labelled ligand promoted by a virtually infinite dilution can still be accelerated by the addition of an excess of cold ligand. (5) Dilution can induce the dissociation, not modify or even paradoxically enhance the association of ligand and binding sites, depending on the stoichiometric scheme.This theory could be useful for determining what is the true stoichiometric scheme describing the interaction between cyclic AMP and protein kinases. It can also account for many data on membrane receptors.     

Molecular basis for coupling the plasma membrane to the actin cytoskeleton during clathrin-mediated endocytosis

Dynamic actin filaments are a crucial component of clathrin-mediated endocytosis when endocytic proteins cannot supply enough energy for vesicle budding. Actin cytoskeleton is thought to provide force for membrane invagination or vesicle scission, but how this force is transmitted to the plasma membrane is not understood. Here we describe the molecular mechanism of plasma membrane–actin cytoskeleton coupling mediated by cooperative action of epsin Ent1 and the HIP1R homolog Sla2 in yeast Saccharomyces cerevisiae. Sla2 anchors Ent1 to a stable endocytic coat by an unforeseen interaction between Sla2’s ANTH and Ent1’s ENTH lipid-binding domains. The ANTH and ENTH domains bind each other in a ligand-dependent manner to provide critical anchoring of both proteins to the membrane. The C-terminal parts of Ent1 and Sla2 bind redundantly to actin filaments via a previously unknown phospho-regulated actin-binding domain in Ent1 and the THATCH domain in Sla2. By the synergistic binding to the membrane and redundant interaction with actin, Ent1 and Sla2 form an essential molecular linker that transmits the force generated by the actin cytoskeleton to the plasma membrane, leading to membrane invagination and vesicle budding.     

Rapid signaling at the plasma membrane by a nuclear receptor for thyroid hormone

Many nuclear hormones have physiological effects that are too rapid to be explained by changes in gene expression and are often attributed to unidentified or novel G protein-coupled receptors. Thyroid hormone is essential for normal human brain development, but the molecular mechanisms responsible for its effects remain to be identified. Here, we present direct molecular evidence for potassium channel stimulation in a rat pituitary cell line (GH(4)C(1)) by a nuclear receptor for thyroid hormone, TRbeta, acting rapidly at the plasma membrane through phosphatidylinositol 3-kinase (PI3K) to slow the deactivation of KCNH2 channels already in the membrane. Signaling was disrupted by heterologous expression of TRbeta receptors with mutations in the ligand-binding domain that are associated with neurological disorders in humans, but not by mutations that disrupt DNA binding. More importantly, PI3K-dependent signaling was reconstituted in cell-free patches of membrane from CHO cells by heterologous expression of human KCNH2 channels and TRbeta, but not TRalpha, receptors. TRbeta signaling through PI3K provides a molecular explanation for the essential role of thyroid hormone in human brain development and adult lipid metabolism.     

A specific role of AGS3 in the surface expression of plasma membrane proteins

Activator of G protein signaling 3 (AGS3), originally identified in a functional screen for mammalian proteins that activate heterotrimeric G protein signaling, is known to be involved in drug-seeking behavior and is up-regulated during cocaine withdrawal in animal models. These observations indicate a potential role for AGS3 in the formation or maintenance of neural plasticity. We have found that the overexpression of AGS3 alters the surface-to-total ratios of a subset of heterologously expressed plasma membrane receptors and channels. Further analysis of the endocytic trafficking of one such protein by a biotin-based internalization assay suggests that overexpression of AGS3 moderately affects the internalization or recycling of surface proteins. Moreover, AGS3 overexpression and siRNA-mediated knockdown of AGS3 both result in the dispersal of two endogenously expressed trans-Golgi network (TGN)-associated cargo proteins without influencing those in the cis- or medial-Golgi compartments. Finally, adding a TGN-localization signal to a CD4-derived reporter renders the trafficking of fusion protein sensitive to AGS3. Taken together, our data support a model wherein AGS3 modulates the protein trafficking along the TGN/plasma membrane/endosome loop.     

A two-component protein condensate of the EGFR cytoplasmic tail and Grb2 regulates Ras activation by SOS at the membrane

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ∼200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and crosslinking through a Grb2-Grb2 binding interface. The Grb2 Y160 residue plays a structurally critical role in the Grb2-Grb2 interaction, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that the condensation state of the EGFR tail controls the ability of SOS, recruited via Grb2, to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

Recently, a class of phenomena known as protein condensation phase transitions has begun to emerge in biology. Originally identified in the context of nuclear organization (1) and gene expression (2), a distinct two-dimensional protein condensation on the cell membrane has now been discovered in the T cell receptor (TCR) signaling system involving the scaffold protein LAT (3–5). TCR activation results in phosphorylation of LAT on at least four distinct tyrosine sites, which subsequently recruit the adaptor protein Grb2 and the signaling molecule PLCγ via selective binding interactions with their SH2 domains. Additional scaffold and signaling molecules, including SOS, GADS, and SLP76, are recruited to Grb2 and PLCγ through further specific protein–protein interactions (6, 7). Multivalency among some of these binding interactions can crosslink LAT molecules in a two-dimensional bond percolation network on the membrane surface. The resulting LAT protein condensate resembles the nephrin:NCK:N-WASP condensate (8) in that both form on the membrane surface under control of tyrosine phosphorylation and exert at least one aspect of functional control over signaling output via a distinct type of kinetic regulatory mechanism (9–11). The basic molecular features controlling the LAT and nephrin protein condensates are common among biological signaling machinery, and other similar condensates continue to be discovered (12, 13). The LAT condensation shares downstream signaling molecules with the EGF-receptor (EGFR) signaling system, raising the question if EGFR may participate in a signaling-mediated protein condensation itself.EGFR signals to the mitogen-activated protein kinase (MAPK) pathway and controls key cellular functions, including growth and proliferation (14–16). EGFR is a paradigmatic model system in studies of signal transduction, and immense, collective scientific effort has revealed the inner workings of its signaling mechanism down to the atomic level (17). EGFR is autoinhibited in its monomeric form. Ligand-driven activation is achieved through formation of an asymmetric receptor dimer in which one kinase activates the other to phosphorylate the nine tyrosine sites in the C-terminal tails (17, 18). There is an obvious conceptual connection between EGFR and the LAT signaling system in T cells. The ∼200-residue–long cytoplasmic tail of EGFR resembles LAT in that both are intrinsically disordered and contain multiple sites of tyrosine phosphorylation that recruit adaptor proteins, including Grb2, upon receptor activation (19). Phosphorylation at tyrosine residues Y1068, Y1086, Y1148, and Y1173 in the EGFR tail creates sites to which Grb2 can bind via its SH2 domain. EGFR-associated Grb2 subsequently recruits SOS, through binding of its SH3 domains to the proline-rich domain of SOS. Once at the membrane, SOS undergoes a multistep autoinhibition-release process and begins to catalyze nucleotide exchange of RasGDP to RasGTP, activating Ras and the MAPK pathway (20).While these most basic elements of the EGFR activation mechanism are widely accepted, larger-scale features of the signaling complex remain enigmatic. A number of studies have reported higher-ordered multimers of EGFR during activation, including early observations by Förster Resonance Energy Transfer and fluorescence lifetime studies (21–23), as have more recent studies using single molecule (24, 25) and computational methods (26). Structural analyses and point mutation studies on EGFR have identified a binding interface enabling EGFR asymmetric dimers to associate (27), but the role of these higher-order assemblies remains unclear. At the same time, many functional properties of the signaling system remain unexplained as well. For example, EGFR is a frequently altered oncogene in human cancers, and drugs (including tyrosine kinase inhibitors) targeting EGFR signaling have produced impressive initial patient responses (28). All too often, however, these drugs fail to offer sustained patient benefits, in large part because of poorly understood resistance mechanisms (29). Physical aspects of the cellular microenvironment have been implicated as possible contributors to resistance development (30), and there is a growing realization that EGFR possesses kinase-independent (e.g., signaling independent) prosurvival functions in cancer cells (31). These points fuel speculation that additional layers of regulation over the EGFR signaling mechanism exist, including at the level of the receptor signaling complex itself.Here we report that EGFR undergoes a protein condensation-phase transition upon activation. We reconstituted the cytoplasmic tails of EGFR on supported bilayers and characterized the system behavior upon interaction with Grb2 and SOS, using total internal reflection fluorescence (TIRF) imaging. This experimental platform has been highly effective for revealing both phase-transition characteristics and functional signaling aspects of LAT protein condensates (4, 5, 10, 32–34). Published reports on the LAT system to date have emphasized SOS (or the SOS proline-rich [PR] domain) as a critical crosslinking element. Titrating the SOS PR domain into an initially homogeneous mixture of phosphorylated LAT and Grb2 revealed a sharp transition to the condensed phase, which we have also observed with the EGFR:Grb2:SOS system. Under slightly different conditions, however, we report observations of an EGFR:Grb2 condensation-phase transition without any SOS or other crosslinking molecule. We show that crosslinking is achieved through a Grb2–Grb2 binding interface. Phosphorylation on Grb2 at Y160 as well as a Y160E mutation [both reported to disrupt Grb2–Grb2 binding (35, 36)] were observed to prevent formation of EGFR condensates. We note that the evidence of Grb2–Grb2 binding we observed occurred in the context of EGFR-associated Grb2, which is localized to the membrane surface; free Grb2 dimers are not necessary.The consequence of EGFR condensation on downstream signaling is characterized by mapping the catalytic efficiency of SOS to activate Ras as a function of the EGFR condensation state. SOS is the primary Ras guanine nucleotide exchange factor (GEF) responsible for activating Ras in the EGFR-to-MAPK signaling pathway (37–40). At the membrane, SOS undergoes a multistep process of autoinhibition release before beginning to activate Ras. Once fully activated, SOS is highly processive, and a single SOS molecule can activate hundreds of Ras molecules before disengaging from the membrane (41–43). Autoinhibition release in SOS is a slow process, which necessitates that SOS be retained at the membrane for an extended time in order for Ras activation to begin (5, 10). This delay between initial recruitment of SOS and subsequent initiation of its Ras GEF activity provides a kinetic proofreading mechanism that essentially requires SOS to achieve multivalent engagement with the membrane (e.g., through multiple Grb2 or other interactions) in order for it to activate any Ras molecules.Experimental results described here reveal that Ras activation by SOS is strongly enhanced by EGFR condensation. Calibrated measurements of both SOS recruitment and Ras activation confirmed enhanced SOS catalytic activity on a per-molecule basis, in addition to enhanced recruitment to the condensates. These results suggest that a Grb2-mediated EGFR protein condensation-phase transition is a functional element controlling signal propagation from EGFR downstream to the MAPK signaling pathway.     

Ras-related protein Rab10 facilitates TLR4 signaling by promoting replenishment of TLR4 onto the plasma membrane

The Toll-like receptor (TLR)4 receptor complex, TLR4/MD-2, plays an important role in the inflammatory response against lipopolysaccharide, a ubiquitous membrane component in Gram-negative bacteria. Ligand recognition by TLR4 initiates multiple intracellular signaling pathways, leading to production of proinflammatory mediators and type I IFN. Ligand interaction also leads to internalization of the surface receptor complex into lysosomes, leading to the degradation of TLR4 and the termination of LPS response. However, surface level of TLR4 receptor complex is maintained via continuous replenishment of TLR4 from intracellular compartments like Golgi and endosomes. Here we show that continuous replenishment of TLR4 from Golgi to plasma membrane is regulated by the small GTPase Rab10, which is essential for optimal macrophage activation following LPS stimulation. Expression of Rab10 is inducible by LPS. Blockade of Rab10 function leads to decreased membrane TLR4 expression and diminished production of inflammatory cytokines and interferons upon LPS stimulation. These findings suggest that Rab10 expression provides a mechanism to refine TLR4 signaling by regulating the trafficking rate of TLR4 onto the plasma membrane. In addition, we show that altered Rab10 expression in macrophages influences disease severity in an in vivo model of LPS-induced acute lung injury, suggesting Rab10 as a possible therapeutic target for human acute respiratory distress syndrome (ARDS).     

Exocytosis occurs at the lateral plasma membrane of the pancreatic acinar cell during supramaximal secretagogue stimulation

In vitro and in vivo studies indicate that the secretory response to both caerulein and carbamylcholine stimulation is biphasic. Over the range of submaximal to maximal concentrations of secretagogues, discharge of exocrine proteins in vitro into the incubation medium and in vivo into the pancreatic duct increased and morphologic analysis indicated that exocytosis of zymogen granules occurred exclusively at the luminal membrane. Under in vivo conditions, supramaximal stimulation with caerulein or carbamylcholine resulted in a dose-dependent decrease in amylase release into the pancreatic duct and increase in the appearance of amylase in the blood circulation. Under in vitro or in vivo conditions, supramaximal secretagogue stimulation resulted in marked inhibition of exocytotic activity at the luminal plasma membrane, the appearance of intergranule contacts and fusions within the cytoplasm, and the appearance of exocytotic activity at the lateral plasma membrane. Lateral exocytotic images were observed with individual and fused zymogen granules and autophagic vacuoles. This redirection in the final step of the secretory pathway provides in part the biological basis for the increased appearance of pancreatic (pro)enzymes in the interstitial fluid and serum during supramaximal secretagogue stimulation.     

Sodium ion-coupled uptake of taurocholate by rat-liver plasma membrane vesicles

ABSTRACT— Uptake of taurocholate into plasma membrane vesicles isolated from rat liver was investigated. In the presence of an extra- to intravesicular gradient of Na+ ions, a typical “overshoot” phenomenon in the accumulation pattern was observed. Osmotic manipulation of the incubation medium indicated that the transport of this bile acid occurs into an osmotically active intravesicular space. Uptake of taurocholate as measured after 1 min was specifically stimulated by Na+ ions: NaNO₃ and NaCl were capable of supporting accumulation, whereas KNO₃ was not. Na+-coupled uptake of taurocholate showed saturation kinetics and was inhibited by other bile acids or by preloading the vesicles with Na+. Our observations support the idea of a carrier-mediated bile-acid uptake system, as suggested previously for the intact rat liver and isolated rat hepatocytes. When the electrical potential difference across the vesicle membrane was changed by inducing different diffusion potentials (anion replacement), a more negative potential inside stimulated Na+-dependent taurocholate transport. The results demonstrate that rat-liver plasma membrane vesicles possess an electrogenic Na+-coupled transport system for taurocholate.     

A mechanism of self-lipid endocytosis mediated by the receptor Mincle

Cellular lipid uptake (through endocytosis) is a basic physiological process. Dysregulation of this process underlies the pathogenesis of diseases such as atherosclerosis, obesity, diabetes, and cancer. However, to date, only some mechanisms of lipid endocytosis have been discovered. Here, we show a previously unknown mechanism of lipid cargo uptake into cells mediated by the receptor Mincle. We found that the receptor Mincle, previously shown to be a pattern recognition receptor of the innate immune system, tightly binds a range of self-lipids. Moreover, we revealed the minimal molecular motif in lipids that is sufficient for Mincle recognition. Superresolution microscopy showed that Mincle forms vesicles in cytoplasm and colocalizes with added fluorescent lipids in endothelial cells but does not colocalize with either clathrin or caveolin-1, and the added lipids were predominantly incorporated in vesicles that expressed Mincle. Using a model of ganglioside GM3 uptake in brain vessel endothelial cells, we show that the knockout of Mincle led to a dramatic decrease in lipid endocytosis. Taken together, our results have revealed a fundamental lipid endocytosis pathway, which we call Mincle-mediated endocytosis (MiME), and indicate a prospective target for the treatment of disorders of lipid metabolism, which are rapidly increasing in prevalence.

Some endocytic pathways in eukaryotic cells have been described, and the major endocytic routes for the internalization of many cargoes are clathrin- and caveolae-mediated endocytosis (1–3). Additionally, some C-type lectins are important players in endocytic processes, including lectin-like oxidized low-density lipoprotein receptor-1, which drives the endocytosis of acetylated low-density lipoproteins (4); thrombomodulin, which drives thrombin internalization (5); and galectin-3, which drives the formation of clathrin-independent carriers for cargo internalization (6). Furthermore, the glycolipid–lectin hypothesis, which explains how cell wall glycolipids and lectins mediate nanoenvironments from which glycosylated cargo proteins endocytose, was recently proposed (1). One of the C-type lectin receptor, Mincle (Clec4e), is considered a pattern recognition receptor of the innate immune system which recognizes both self-lipids and non-self-lipids (7, 8). Early studies showed that Mincle recognizes bacterial and fungal glycolipids (9, 10). Additionally, Mincle recognizes endogenous cholesterol (11) and cholesterol sulfate (12), inducing an inflammatory response. Recently, one of the structurally simplest glycosphingolipids (GSLs), β-glucosylceramides, was shown to bind Mincle on myeloid cells and induce immediate inflammatory responses (13). However, to date Mincle’s role in lipid uptake is unclear. As Mincle is a pattern recognition receptor, we hypothesized that it may recognize a broad range of lipids with similar structure and may play role in lipid endocytosis.     

Plasma membrane insertion of the AMPA receptor GluA2 subunit is regulated by NSF binding and Q/R editing of the ion pore

The delivery of AMPA receptors to the plasma membrane is a critical step both for the synaptic delivery of these receptors and for the regulation of synaptic transmission. To directly visualize fusion events of transport vesicles containing the AMPA receptor GluA2 subunit with the plasma membrane we used pHluorin-tagged GluA2 subunits and total internal reflection fluorescence microscopy. We demonstrate that the plasma membrane insertion of GluA2 requires the NSF binding site within its intracellular cytoplasmic domain and that RNA editing of the Q/R site in the ion channel region plays a key role in GluA2 plasma membrane insertion. Finally, we show that plasma membrane insertion of heteromeric GluA2/3 receptors follows the same rules as homomeric GluA2 receptors. These results demonstrate that the plasma membrane delivery of GluA2 containing AMPA receptors is regulated by its unique structural elements.     

A 75-kDa polypeptide, located primarily at the plasma membrane of carrot cell-suspension cultures, is photoaffinity labeled by the calcium channel blocker LU 49888

Calcium channel blockers of the phenylalkylamine family bind specifically to membranes and inhibit calcium uptake in carrot protoplast. LU 49888, an azido derivative of phenylalkylamine, behaves as its unmodified homolog in terms of affinity and specificity and therefore allows us to probe the receptor by photoaffinity labeling. Upon UV irradiation, a 75-kDa peptide was specifically labeled. Incubation of microsomes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, a zwitterionic detergent, led to the solubilization of the LU 49888-binding protein. Electrophoretic analysis under denaturing conditions and gel filtration of the solubilized "receptor-ligand" complex show a 75-kDa peptide mainly located at the plasma membrane. Consequently the LU 49888-binding protein in plants differs significantly from its animal counterpart by its size and may be a primary target for external signal molecules.     

Forward transport of proteins in the plasma membrane of migrating cerebellar granule cells

Significance

Newly differentiated neurons migrate over long distances to reach their proper destination in the developing brain. The mechanisms by which neurons accomplish this translocation remain to be clarified. By analyzing the trajectories of antibody-coated single quantum dots bound to specific plasma membrane proteins, we found that membrane proteins of migrating cultured cerebellar granule cells exhibited net forward translocation in a form of biased drift, which is superimposed upon Brownian motion, and that this biased drift appears to be driven by myosin II-dependent active transport processes. Thus, plasma membrane proteins undergo forward translocation in unison with cytoplasmic components in migrating neurons.     

cAMP regulates DEP domain-mediated binding of the guanine nucleotide exchange factor Epac1 to phosphatidic acid at the plasma membrane

Epac1 is a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap. Upon cAMP binding, Epac1 undergoes a conformational change that results in its release from autoinhibition. In addition, cAMP induces the translocation of Epac1 from the cytosol to the plasma membrane. This relocalization of Epac1 is required for efficient activation of plasma membrane-located Rap and for cAMP-induced cell adhesion. This translocation requires the Dishevelled, Egl-10, Pleckstrin (DEP) domain, but the molecular entity that serves as the plasma membrane anchor and the possible mechanism of regulated binding remains elusive. Here we show that Epac1 binds directly to phosphatidic acid. Similar to the cAMP-induced Epac1 translocation, this binding is regulated by cAMP and requires the DEP domain. Furthermore, depletion of phosphatidic acid by inhibition of phospholipase D1 prevents cAMP-induced translocation of Epac1 as well as the subsequent activation of Rap at the plasma membrane. Finally, mutation of a single basic residue within a polybasic stretch of the DEP domain, which abolishes translocation, also prevents binding to phosphatidic acid. From these results we conclude that cAMP induces a conformational change in Epac1 that enables DEP domain-mediated binding to phosphatidic acid, resulting in the tethering of Epac1 at the plasma membrane and subsequent activation of Rap.     

ErbB2 receptor controls microtubule capture by recruiting ACF7 to the plasma membrane of migrating cells

Microtubules (MTs) contribute to key processes during cell motility, including the regulation of focal adhesion turnover and the establishment and maintenance of cell orientation. It was previously demonstrated that the ErbB2 receptor tyrosine kinase regulated MT outgrowth to the cell cortex via a complex including Memo, the GTPase RhoA, and the formin mDia1. But the mechanism that linked this signaling module to MTs remained undefined. We report that ErbB2-induced repression of glycogen synthase kinase-3 (GSK3) activity, mediated by Memo and mDia1, is required for MT capture and stabilization. Memo-dependent inhibition of GSK3 allows the relocalization of APC (adenomatous polyposis coli) and cytoplasmic linker-associated protein 2 (CLASP2), known MT-associated proteins, to the plasma membrane and ruffles. Peripheral microtubule extension also requires expression of the plus-end binding protein EB1 and its recently described interactor, the spectraplakin ACF7. In fact, in migrating cells, ACF7 localizes to the plasma membrane and ruffles, in a Memo-, GSK3-, and APC-dependent manner. Finally, we demonstrate that ACF7 targeting to the plasma membrane is both required and sufficient for MT capture downstream of ErbB2. This function of ACF7 does not require its recently described ATPase activity. By defining the signaling pathway by which ErbB2 allows MT capture and stabilization at the cell leading edge, we provide insights into the mechanism underlying cell motility and steering.