Natasha K. Hussain

Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.

Signaling on the Endocytic Pathway

Ligand binding to receptor tyrosine kinases and G‐protein‐coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin‐coated pits and vesicles. While receptor‐mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand‐activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal‐regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin‐dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways.

ENTH/ANTH proteins and clathrin-mediated membrane budding

The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated endocytosis. Structural analyses and ligand-binding studies have shown that a set of proteins previously designated as harboring an ENTH domain in fact contain a highly similar, yet unique module referred to as an AP180 N-terminal homology (ANTH) domain. ENTH and ANTH (E/ANTH) domains bind both inositol phospholipids and proteins and contribute to the nucleation and formation of clathrin coats on membranes. ENTH domains also function in the development of membrane curvature through lipid remodeling during the formation of clathrin-coated vesicles. E/ANTH-bearing proteins have recently been shown to function with adaptor protein-1 and GGA adaptors at the trans-Golgi network, which suggests that E/ANTH domains are universal components of the machinery for clathrin-mediated membrane budding.

Splice Variants of Intersectin Are Components of the Endocytic Machinery in Neurons and Nonneuronal Cells

We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrin-coated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is co-localized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain

We recently identified intersectin, a protein containing two EH and five SH3 domains, as a component of the endocytic machinery. The N‐terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin‐coated pits. We have now identified a brain‐enriched, 170 kDa protein (p170) that interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR, and the 170 kDa mammalian son‐of‐sevenless (mSos1) protein, a guanine‐nucleotide exchange factor for Ras, contains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co‐enriched in nerve terminals and are co‐immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin–mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor‐mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes.

Retrograde signaling by the neurotrophins follows a well-worn trk

The mechanism that allows a neuron to send cues received at its terminal to its cell body and nucleus has proved elusive. However, a recent study by Howe and colleagues indicates that neurotrophin signaling via the trkA receptor requires formation of a signaling endosome containing NGF and trkA. Thus, endocytosis of the neurotrophin-receptor complex is a crucial step in the generation of intracellular signaling platforms required for activation and compartmentalization of signaling events.

S-nitrosylation of AMPA receptor GluA1 regulates phosphorylation, single-channel conductance, and endocytosis

NMDA receptor activation can elicit synaptic plasticity by augmenting conductance of the AMPA receptor GluA1 subsequent to phosphorylation at S831 by Ca2+-dependent kinases. NMDA receptor activation also regulates synaptic plasticity by causing endocytosis of AMPA receptor GluA1. We demonstrate a unique signaling cascade for these processes mediated by NMDA receptor-dependent NO formation and GluA1 S-nitrosylation. Thus, S-nitrosylation of GluA1 at C875 enhances S831 phosphorylation, facilitates the associated AMPA receptor conductance increase, and results in endocytosis by increasing receptor binding to the AP2 protein of the endocytotic machinery.

MINK and TNIK Differentially Act on Rap2-Mediated Signal Transduction to Regulate Neuronal Structure and AMPA Receptor Function

Misshapen/NIKs (Nck-interacting kinases)-related kinase (MINK) and closely related TRAF2/Nck-interacting kinase (TNIK) are proteins that specifically bind to activated Rap2 and are thus hypothesized to relay its downstream signal transduction. Activated Rap2 has been found to stimulate dendritic pruning, reduce synaptic density and cause removal of synaptic AMPA receptors (AMPA-Rs) (Zhu et al., 2005; Fu et al., 2007). Here we report that MINK and TNIK are postsynaptically enriched proteins whose clustering within dendrites is bidirectionally regulated by the activation state of Rap2. Expression of MINK and TNIK in neurons is required for normal dendritic arborization and surface expression of AMPA receptors. Overexpression of a truncated MINK mutant unable to interact with Rap2 leads to reduced dendritic branching and this MINK-mediated effect on neuronal morphology is dependent upon Rap2 activation. While similarly truncated TNIK also reduces neuronal complexity, its effect does not require Rap2 activity. Furthermore, Rap2-mediated removal of surface AMPA-Rs from spines is entirely abrogated by coexpression of MINK, but not TNIK. Thus, although both MINK and TNIK bind GTP-bound Rap2, these kinases employ distinct mechanisms to modulate Rap2-mediated signaling. MINK appears to antagonize Rap2 signal transduction by binding to activated Rap2. We suggest that MINK interaction with Rap2 plays a critical role in maintaining the morphological integrity of dendrites and synaptic transmission.

Sorting Nexin 27 regulates basal and activity-dependent trafficking of AMPARs

Significance AMPA-type glutamate receptors (AMPARs) are principal regulators of synaptic signaling in the brain. Modulation of AMPA receptor activity, whether through changes in surface expression or conductance, contributes significantly to the dynamic nature of neuronal networks. AMPA receptor mediated-synaptic plasticity is thought to underlie learning and memory, as aberrant AMPAR trafficking contributes to impaired plasticity and to memory deficits. We demonstrate that the membrane trafficking and sorting protein, SNX27, regulates basal and activity dependent targeting of AMPA receptors to the neuronal surface. Elucidating the mechanism of receptor sorting mediated by SNX27 will not only further our understanding AMPAR-mediated changes in synaptic plasticity, but may ultimately shed light on the molecular mechanisms that govern learning. Activity-dependent changes in synaptic strength have long been postulated as cellular correlates of learning and memory. Long-term potentiation (LTP), a well characterized form of synaptic plasticity, is often expressed as an increase in the number of postsynaptic AMPA-type glutamate receptors (AMPARs). Although the precise molecular mechanisms governing LTP remain elusive, this study identifies one member of the sorting nexin family, Sorting Nexin 27 (SNX27), as a critical component in this process. The ability of sorting nexins to bind specific phospholipids as well as their propensity to form protein–protein complexes, points to a role for these proteins in membrane trafficking and protein sorting. Here, we demonstrate that SNX27 binds to AMPARs, and that this interaction is regulated in an activity-dependent manner. Furthermore, we provide evidence that SNX27 is synaptically enriched and its level of expression regulates targeting of AMPARs to the neuronal surface. Loss of SNX27 abolishes recruitment of surface AMPARs during chemical LTP. Collectively, our data suggest a role for SNX27 in modulating synaptic plasticity through regulated interaction with AMPARs.