The Minimal Autoinhibited Unit of the Guanine Nucleotide Exchange Factor Intersectin

Intersectin-1L is a member of the Dbl homology (DH) domain guanine nucleotide exchange factors (GEF) which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E)) being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E)-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E)-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain.     

Autoinhibition of GEF activity in intersectin 1 is mediated by the short SH3-DH domain linker

Intersectin 1L (ITSN1L) acts as a specific guanine nucleotide exchange factor (GEF) for the small guanine nucleotide binding protein Cdc42 via its C‐terminal DH domain. Interestingly, constructs of ITSN1L that comprise additional domains, for instance the five SH3 domains amino‐terminal of the DH domain, were shown to be inhibited in their exchange factor activity. Here, we investigate the inhibitory mechanism of ITSN1L in detail and identify a novel short amino acid motif which mediates autoinhibition. We found this motif to be located in the linker region between the SH3 domains and the DH domain, and we show that within this motif W1221 acts as key residue in establishing the inhibitory interaction. This assigns ITSN1L to a growing class of GEFs that are regulated by a short amino acid motif inhibiting GEF activity by an intramolecular interaction. Moreover, we quantify the interaction between the ITSN1L SH3 domains and the Cdc42 effector N‐WASP using fluorescence anisotropy binding experiments. As the SH3 domains are not involved in autoinhibition, binding of N‐WASP does not release inhibition of nucleotide exchange activity in kinetic experiments, in contrast to earlier observations.     

The activity of the GTPase-activating protein CdGAP is regulated by the endocytic protein intersectin.

The Rho GTPases RhoA, Rac1, and Cdc42 play a major role in regulating the reorganization of the actin cytoskeleton. We recently identified CdGAP, a novel GTPase-activating protein with activity toward Rac1 and Cdc42. CdGAP consists of a N-terminal GAP domain, a central domain, and a C-terminal proline-rich domain. Here we show that through a subset of its Src homology 3 domains, the endocytic protein intersectin interacts with CdGAP. In platelet-derived growth factor-stimulated Swiss 3T3 cells, intersectin co-localizes with CdGAP and inhibits its GAP activity toward Rac1. Intersectin-Src homology 3 also inhibits CdGAP activity in GAP assays in vitro. Although the C-terminal proline-rich domain of CdGAP is required for the regulation of its GAP activity by intersectin both in vivo and in vitro, it is not necessary for CdGAP-intersectin interaction. Our data suggest that the central domain of CdGAP is required for CdGAP-intersectin interaction. Thus, we propose a model in which intersectin binding results in a change of CdGAP conformation involving the proline-rich domain that leads to the inhibition of its GAP activity. These observations provide the first demonstration of a direct regulation of RhoGAP activity through a protein-protein interaction and suggest a function for intersectin in Rac1 regulation and actin dynamics.     

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, con- tains 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.     

Intersectin can regulate the Ras/MAP kinase pathway independent of its role in endocytosis

We previously identified intersectin, a multiple EH and SH3 domain-containing protein, as a component of the endocytic machinery. Overexpression of the SH3 domains of intersectin blocks transferrin receptor endocytosis, possibly by disrupting targeting of accessory proteins of clathrin-coated pit formation. More recently, we identified mammalian Sos, a guanine-nucleotide exchange factor for Ras, as an intersectin SH3 domain-binding partner. We now demonstrate that overexpression of intersectin's SH3 domains blocks activation of Ras and MAP kinase in various cell lines. Several studies suggest that activation of MAP kinase downstream of multiple receptor types is dependent on endocytosis. Thus, the dominant-negative effect of the SH3 domains on Ras/MAP kinase activation may be indirectly mediated through a block in endocytosis. Consistent with this idea, incubating cells at 4 degrees C or with phenylarsine oxide, treatments previously established to inhibit EGF receptor endocytosis, blocks EGF-dependent activation of MAP kinase. However, under these conditions, Ras activity is unaffected and overexpression of the SH3 domains of intersectin is still able to block Ras activation. Thus, intersectin SH3 domain overexpression can effect EGF-mediated MAP kinase activation directly through a block in Ras, consistent with a functional role for intersectin in Ras activation.     

Cdc42 and Ras cooperate to mediate cellular transformation by intersectin-L

Cdc42, a Ras-related GTP-binding protein, has been implicated in the regulation of the actin cytoskeleton, membrane trafficking, cell-cycle progression, and malignant transformation. We have shown previously that a Cdc42 mutant (Cdc42(F28L)), capable of spontaneously exchanging GDP for GTP (referred to as "fast-cycling"), transformed NIH 3T3 cells because of its ability to interfere with epidermal growth factor receptor (EGFR)-Cbl interactions and EGFR down-regulation. To further examine the link between the hyperactivation of Cdc42 and its ability to alter EGFR signaling and thereby cause cellular transformation, we examined the effects of expressing different forms of the Cdc42-specific guanine nucleotide exchange factor, intersectin-L, in fibroblasts. Full-length intersectin-L exhibited little ability to stimulate nucleotide exchange on Cdc42, whereas a truncated version that contained five Src homology 3 (SH3) domains, the Dbl and pleckstrin homology domains (DH and PH domains, respectively), and a C2 domain (designated as SH3A-C2) showed modest guanine nucleotide exchange factor activity, whereas a form containing just the DH, PH, and C2 domains (DH-C2) strongly activated Cdc42. However, DH-C2 showed little ability to stimulate growth in low serum or colony formation in soft agar, whereas SH3A-C2 gave rise to a much stronger stimulation of cell growth in low serum and was highly effective in stimulating colony formation. Moreover, although SH3A-C2 strongly transformed fibroblasts, it differed from the actions of the Cdc42(F28L) mutant, as SH3A-C2 showed little ability to alter EGFR levels or the lifetime of EGF-coupled signaling through ERK. Rather, we found that SH3A-C2 exhibited strong transforming activity through its ability to mediate cooperation between Ras and Cdc42.     

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.     

Investigation of the Interaction between Cdc42 and Its Effector TOCA1: HANDOVER OF Cdc42 TO THE ACTIN REGULATOR N-WASP IS FACILITATED BY DIFFERENTIAL BINDING AFFINITIES*

Transducer of Cdc42-dependent actin assembly protein 1 (TOCA1) is an effector of the Rho family small G protein Cdc42. It contains a membrane-deforming F-BAR domain as well as a Src homology 3 (SH3) domain and a G protein-binding homology region 1 (HR1) domain. TOCA1 binding to Cdc42 leads to actin rearrangements, which are thought to be involved in processes such as endocytosis, filopodia formation, and cell migration. We have solved the structure of the HR1 domain of TOCA1, providing the first structural data for this protein. We have found that the TOCA1 HR1, like the closely related CIP4 HR1, has interesting structural features that are not observed in other HR1 domains. We have also investigated the binding of the TOCA HR1 domain to Cdc42 and the potential ternary complex between Cdc42 and the G protein-binding regions of TOCA1 and a member of the Wiskott-Aldrich syndrome protein family, N-WASP. TOCA1 binds Cdc42 with micromolar affinity, in contrast to the nanomolar affinity of the N-WASP G protein-binding region for Cdc42. NMR experiments show that the Cdc42-binding domain from N-WASP is able to displace TOCA1 HR1 from Cdc42, whereas the N-WASP domain but not the TOCA1 HR1 domain inhibits actin polymerization. This suggests that TOCA1 binding to Cdc42 is an early step in the Cdc42-dependent pathways that govern actin dynamics, and the differential binding affinities of the effectors facilitate a handover from TOCA1 to N-WASP, which can then drive recruitment of the actin-modifying machinery.     

Pleiotropic function of intersectin homologue Cin1 in Cryptococcus neoformans

The manifestation of virulence traits in Cryptococcus neoformans is thought to rely on intracellular transport, a process not fully explored in this pathogenic fungus. Through interaction cloning, we identified a multi‐modular protein, Cin1 (cryptococcal intersectin 1), whose domain structure is similar to that of the human endocytic protein ITSN1. Cin1 contains an N‐terminal EH domain, a central coiled‐coil region, a WH2 domain, two SH3 domains and a C‐terminal RhoGEF (DH)‐PH domain. Interestingly, alternative mRNA splicing resulted in two Cin1 isoforms, and Cin1 homologues are also restricted to basidiomycetous fungi. Disruption of the CIN1 gene had a pleiotropic effect on growth, normal cytokinesis, intracellular transports and the production of several virulence factors. Additionally, Cin1 interacts with cryptococcal Cdc42 and Wsp1 (a WASP homologue) proteins in vitro, suggesting a conserved role in the regulation of the actin cytoskeleton. However, deletion of RhoGEF or SH3 and RhoGEF domains did not result in any phenotypic changes, suggesting that functional redundancy exists in proteins containing similar domains or that the activities by other domains are necessary for Cin1 function. Our study presents the first evidence of a multi‐modular protein whose function in intracellular transport underlies the growth, differentiation and virulence of a pathogenic microorganism.     

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.     

Modulation of oncogenic DBL activity by phosphoinositol phosphate binding to pleckstrin homology domain

The Dbl family guanine nucleotide exchange factors (GEFs) contain a region of sequence similarity consisting of a catalytic Dbl homology (DH) domain in tandem with a pleckstrin homology (PH) domain. PH domains are involved in the regulated targeting of signaling molecules to plasma membranes by protein-protein and/or protein-lipid interactions. Here we show that Dbl PH domain binding to phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate results in the inhibition of Dbl GEF activity on Rho family GTPase Cdc42. Phosphatidylinositol 4,5-bisphosphate binding to the PH domain significantly inhibits the Cdc42 interactive activity of the DH domain suggesting that the DH domain is subjected to the PH domain modulation under the influence of phosphoinositides (PIPs). We generated Dbl mutants unable to interact with PIPs. These mutants retained GEF activity on Cdc42 in the presence of PIPs and showed a markedly enhanced activating potential for both Cdc42 and RhoA in vivo while displaying decreased cellular transforming activity. Immunofluorescence analysis of NIH3T3 transfectants revealed that whereas the PH domain localizes to actin stress fibers and plasma membrane, the PH mutants are no longer detectable on the plasma membrane. These results suggest that modulation of PIPs in both the GEF catalytic activity and the targeting to plasma membrane determines the outcome of the biologic activity of Dbl.     

Identification and characterization of a novel Rho GTPase activating protein implicated in receptor-mediated endocytosis

Cbl-interacting protein of 85 kDa (CIN85) is a recently identified adaptor protein involved in the endocytic process of several receptor tyrosine kinases. Here we have identified a novel RhoGAP, CIN85 associated multi-domain containing Rho1 (CAMGAP1) as a binding protein for CIN85. CAMGAP1 is composed of an Src homology 3 (SH3) domain, multiple WW domains, a proline-rich region, a PH domain and a RhoGAP domain, and has the domain architecture similar to ARHGAP9 and ARHGAP12. CAMGAP1 mRNA is widely distributed in murine tissues. Biochemical assays showed its GAP activity toward Rac1 and Cdc42. Protein binding and expression studies indicated that the second SH3 domain of CIN85 binds to a proline-rich region of CAMGAP1. Overexpression of a truncated form of CAMGAP1 interferes with the internalization of transferrin receptors, suggesting that CAMGAP1 may play a role in clathrin-mediated endocytosis.     

Intersectin-2L Regulates Caveola Endocytosis Secondary to Cdc42-mediated Actin Polymerization

Here we addressed the role of intersectin-2L (ITSN-2L), a guanine nucleotide exchange factor for the Rho GTPase Cdc42, in the mechanism of caveola endocytosis in endothelial cells (ECs). Immunoprecipitation and co-localization studies showed that ITSN-2L associates with members of the Cdc42-WASp-Arp2/3 actin polymerization pathway. Expression of Dbl homology-pleckstrin homology (DH-PH) region of ITSN-2L (DH-PH_ITSN-2L) induced specific activation of Cdc42, resulting in formation of extensive filopodia, enhanced cortical actin, as well as a shift from G-actin to F-actin. The “catalytically dead” DH-PH domain reversed these effects and induced significant stress fiber formation, without a detectable shift in actin pools. A biotin assay for caveola internalization indicated a significant decrease in the uptake of biotinylated proteins in DH-PH_ITSN-2L-transfected cells compared with control and 1 μm jasplakinolide-treated cells. ECs depleted of ITSN-2L by small interfering RNA, however, showed decreased Cdc42 activation and actin remodeling similar to the defective DH-PH, resulting in 62% increase in caveola-mediated uptake compared with controls. Thus, ITSN-2L, a guanine nucleotide exchange factor for Cdc42, regulates different steps of caveola endocytosis in ECs by controlling the temporal and spatial actin polymerization and remodeling sub-adjacent to the plasma membrane.The polymerization of actin has a central role in clathrin- and caveola-mediated endocytosis (1). Studies have shown a number of protein-protein interactions that suggest a functional relationship between the actin cytoskeleton and endocytic machinery; however, the underlying mechanisms remain unclear. ITSN-2L,² a multifunctional domain protein with two Epsin 15 homology domains, a central coiled-coil region followed by five consecutive Src homology 3 domains, a Dbl homology (DH), a pleckstrin homology (PH), and finally a C2 domain, interacts via the Src homology 3 region with the ubiquitously expressed neural Wiskott-Aldrich syndrome protein (N-WASP) that stimulates actin nucleation through Arp 2/3 complex activation (2). ITSN-2L interaction with N-WASP in turn induces activation of N-WASP in a Cdc42-dependent manner (2, 3). In this way, ITSN-2L on the basis of its DH domain acts as a GEF for the small Rho GTPase Cdc42, similar to its neuronal counterpart ITSN-1L (2, 4). The DH domain of ITSN-2L shows high sequence homology with the corresponding region of ITSN-1L (5), and it possesses all the amino acid residues required for its GEF enzymatic activity (6). Both long ITSN isoforms display immediately distal to the DH domain a PH domain, which may thereby modulate the intrinsic catalytic activity of the DH region (6–8). It has been shown that the PH domain enhances up to 100-fold the DH catalytic activity for some Dbl proteins compared with that measured for DH alone in vitro, whereas for other Dbl proteins the presence of the PH domain negatively regulates GEF activity of the DH region (9). This latter function is apparently mediated by interactions with phosphoinositides (7, 9). However, the PH sequence was shown to be dispensable for GEF activity of ITSN-2L in vitro, but it enhanced the ability to activate Cdc42 in vivo (9). Despite high sequence conservation among Rho GTPases, long ITSN isoforms apparently induce selective activation of Cdc42 due to the overall increasing size of the specificity residues of the GTPases (Cdc42 < Rac1 < RhoA) and the inability of ITSN to accommodate in an analogous position the larger size amino acid chains found in Rac1 and RhoA (10).ITSN-2L, like its alternatively spliced short isoform, is widely expressed in human tissue, and it shows subcellular distribution similar to components of the endocytic machinery (5). In COS-7 cells overexpressing ITSN-2 isoforms, clathrin-mediated transferrin uptake was blocked, consistent with their involvement in the regulation of clathrin-mediated endocytosis (5). By contrast, ITSN-2L overexpression in Jurkat cells stimulated T cell antigen receptor (TCR) internalization, whereas truncated ITSN-2L, deleted for the DH domain, caused significant inhibition of TCR internalization (2). The stimulatory effect of ITSN-2L on TCR endocytosis may be secondary to the ability of ITSN-2L to bind through its Src homology 3 domains the proline-rich domain of N-WASP followed by Cdc42-mediated actin polymerization (2). Although more work is needed to clarify these inconsistencies, both of these studies suggest that ITSN-2L may regulate endocytosis and function cooperatively with N-WASP and Cdc42 to link WASP-mediated actin polymerization to the endocytic machinery (2).Live cultured fibroblast imaging showed that actin polymerization as regulated by the WASP-Arp2/3 complex participates in the late stage of clathrin-mediated endocytosis (11). Therefore, we reasoned that ITSN-2L, as a specific activator of Cdc42, may be essential for actin cytoskeleton polymerization and caveola internalization in ECs. ECs are particularly rich in caveola, and caveola-mediated endocytosis is a fundamental step in mediating the transcytosis of proteins (12, 13), but the mechanisms of caveola-mediated endocytosis and the essential proteins involved remain enigmatic. In this study, we addressed the role of ITSN-2L in the mechanism of caveola internalization in ECs. Our data employing morphological, biochemical, and functional approaches show that ITSN-2L on the basis of Cdc42-mediated spatial actin polymerization is required in the mechanism of caveola-mediated endocytosis.     

Wsp1, a GBD/CRIB domain-containing WASP homolog, is required for growth, morphogenesis, and virulence of Cryptococcus neoformans

Human endocytic protein ITSN1 regulates actin reorganization by activating Rho family GTPases, such as Cdc42. The process is enhanced by ITSN binding of WASP, an effector of Cdc42 and a potent activator of actin polymerization. In the human pathogen Cryptococcus neoformans, endocytic protein Cin1 also interacts with Cdc42 and Wsp1, an uncharacterized WASP homolog, but the significance of these interactions remains unknown. Wsp1 contains several conserved domains, including a WASP homology 1 domain (WH1), a GTPase binding/Cdc42 and Rac interactive binding domain (GBD/CRIB), and a C-terminal domain composed of verprolin-like, central, and acidic motifs (VCA). Thus, Wsp1 exhibits domain compositions more similar to human WASP proteins than Saccharomyces cerevisiae Las17/Bee1, a WASP homolog lacking the GDB/CRIB domain. Wsp1 is not an essential protein; however, the wsp1 mutant exhibited defects in growth, cytokinesis, chitin distribution, and endocytosis and exocytosis. The wsp1 mutant was also unable to undergo genetic cross, produce the polysaccharide capsule, or secrete the enzyme urease. An in vitro phagocytosis assay showed a higher phagocytic index for the wsp1 mutant, whose ability to cause lethal infection in a murine model of cryptococcosis was also attenuated. Our studies reveal divergent evolution of WASP proteins in the fungal phylum and suggest that the conserved function of WASP proteins in the actin cytoskeleton may also impact fungal virulence.     

Function of intersectin in endocytosis and exocytosis

Intersectin is an evolutionarily conserved multifunctional adaptor protein with multifunctional domains. These domains interact with components of the endocytic and exocytic pathways, such as the clathrin mediating synaptic vesicle recycling, the protein related to endocytosis via caveolae, the with-no-lysine kinases related to the regulation of renal outer medullar potassium, and the Cdc42 mediating exocytic pathway. Recently, the understanding of intersectin function in the pathogenesis of endocrine tumor and many neurodegenerative diseases such as Down syndrome, Alzheimer disease has been deepened. This article reviewed the structure and roles in endocytosis/exocytosis and diseases of intersectin.     

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.     

Cdc42 GTPase-activating protein (CdGAP) interacts with the SH3D domain of Intersectin through a novel basic-rich motif

The small GTPases Rac1 and Cdc42 are key regulators of the cytoskeleton. We have previously identified the endocytic protein Intersectin as a binding partner and regulator of Cdc42 GTPase-activating protein (CdGAP) with activity towards Rac1 and Cdc42. This interaction is mediated through the SH3D domain of Intersectin and the central domain of CdGAP, which does not contain any typical proline-rich domain or known SH3-binding motif. Here, we have characterized the Intersectin-SH3D/CdGAP interaction. We show that Intersectin-SH3D interacts directly with a small region of CdGAP highly enriched in basic residues and comprising a novel conserved xKx(K/R)K motif.     

EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25. A protein connection between exocytosis and endocytosis?

In yeast two-hybrid screens for proteins that bind to SNAP-25 and may be involved in exocytosis, we isolated a protein called EHSH1 (for EH domain/SH3 domain-containing protein). Cloning of full-length cDNAs revealed that EHSH1 is composed of an N-terminal region with two EH domains, a central region that is enriched in lysine, leucine, glutamate, arginine, and glutamine (KLERQ domain), and a C-terminal region comprised of five SH3 domains. The third SH3 domain is alternatively spliced. Data bank searches demonstrated that EHSH1 is very similar to Xenopus and human intersectins and to human SH3P17. In addition, we identified expressed sequence tags that encode a second isoform of EHSH1, called EHSH2. EHSH1 is abundantly expressed in brain and at lower levels in all other tissues tested. In binding studies, we found that the central KLERQ domain of EHSH1 binds to recombinant or native brain SNAP-25 and SNAP-23. The C-terminal SH3 domains, by contrast, quantitatively interact with dynamin, a protein involved in endocytosis. Dynamin strongly binds to the alternatively spliced central SH3 domain (SH3C) and the two C-terminal SH3 domains (SH3D and SH3E) but not to the N-terminal SH3 domains (SH3A and SH3B). Immunoprecipitations confirmed that both dynamin and SNAP-25 are complexed to EHSH1 in brain. Our data suggest that EHSH1/intersectin may be a novel adaptor protein that couples endocytic membrane traffic to exocytosis. The ability of multiple SH3 domains in EHSH1 to bind to dynamin suggests that EHSH1 can cluster several dynamin molecules in a manner that is regulated by alternative splicing.     

Cholesterol-sensitive Cdc42 activation regulates actin polymerization for endocytosis via the GEEC pathway

Glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present at the surface of living cells in cholesterol dependent nanoscale clusters. These clusters appear to act as sorting signals for the selective endocytosis of GPI-APs via a Cdc42-regulated, dynamin and clathrin-independent pinocytic pathway called the GPI-AP-enriched early endosomal compartments (GEECs) pathway. Here we show that endocytosis via the GEECs pathway is inhibited by mild depletion of cholesterol, perturbation of actin polymerization or overexpression of the Cdc42/Rac-interactive-binding (CRIB) motif of neural Wiskott-Aldrich syndrome protein (N-WASP). Consistent with the involvement of Cdc42-based actin nanomachinery, nascent endocytic vesicles containing cargo for the GEEC pathway co-localize with fluorescent protein-tagged isoforms of Cdc42, CRIB domain, N-WASP and actin; high-resolution electron microscopy on plasma membrane sheets reveals Cdc42-labelled regions rich in green fluorescent protein-GPI. Using total internal reflection fluorescence microscopy at the single-molecule scale, we find that mild cholesterol depletion alters the dynamics of actin polymerization at the cell surface by inhibiting Cdc42 activation and consequently its stabilization at the cell surface. These results suggest that endocytosis into GEECs occurs through a cholesterol-sensitive, Cdc42-based recruitment of the actin polymerization machinery.