The Rab5 effector EEA1 interacts directly with syntaxin-6.

The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.     

Sequential roles for phosphatidylinositol 3-phosphate and Rab5 in tethering and fusion of early endosomes via their interaction with EEA1

Early endosome antigen 1 (EEA1) is a 170-kDa polypeptide required for endosome fusion in mammalian cells. The COOH terminus of EEA1 contains a FYVE domain that interacts specifically with phosphatidylinositol 3-phosphate (PtdIns-3-P) and a Rab5 GTPase binding region adjacent to the FYVE domain. The dual interaction of EEA1 with both PtdIns-3-P and Rab5 has been hypothesized to provide the specificity required to target EEA1 to early endosomes. To test this hypothesis, we generated truncated (amino acids 1277--1411) and full-length EEA1 constructs containing point mutations in the COOH terminus that impair Rab5 but not PtdIns-3-P binding. These constructs localized to endosomes in intact cells as efficiently as their wild-type counterparts. Furthermore, overexpression of the truncated constructs, both wild-type and mutated, impaired the function of endogenous EEA1 resulting in the accumulation of small, untethered endosomes. These results suggest that association with Rab5 is not necessary for the initial binding and tethering functions of EEA1. A role for Rab5 binding was revealed, however, upon comparison of endosomes in cells expressing full-length wild-type or mutated EEA1. The mutant full-length EEA1 caused the accumulation of endosome clusters and suppressed the enlargement of endosomes caused by a persistently active form of Rab5 (Rab5Q79L). In contrast, expression of wild-type EEA1 with Rab5Q79L enhanced this enlargement. Thus, endosome tethering depends on the interaction of EEA1 with PtdIns-3-P, and its interaction with Rab5 appears to regulate subsequent fusion.     

Direct interaction of Rab4 with syntaxin 4

In the present study, we examined the possible interaction between Rab4 and syntaxin 4, both having been implicated in insulin-induced GLUT4 translocation. Rab4 and syntaxin 4 were coimmunoprecipitated from the lysates of electrically permeabilized rat adipocytes. The interaction between the two proteins was reduced by insulin treatment and increased by the addition of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). An in vitro binding assay revealed that the bacterially expressed Rab4 was bound to a glutathione S-transferase fusion protein containing the cytoplasmic domain of syntaxin 4 (GST-syntaxin 4-(1-273)) but not to syntaxin 1A or vesicle-associated membrane protein-2. The interaction between Rab4 and syntaxin 4 seemed to be regulated by the guanine nucleotide status of Rab4, because 1) GTPgammaS treatment of the cells significantly increased, but guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) treatment decreased the amount of Rab4 pulled down with GST-syntaxin 4-(1-273) from the cell lysates; 2) GTPgammaS loading on Rab4 caused a marked increase in the affinity of Rab4 to syntaxin 4 whereas GDPbetaS loading had little effect; and 3) a GTPase-deficient mutant of Rab4 (Rab4(Q67L)), but not a GTP-binding-defective mutant (Rab4(S22N)), was bound to GST-syntaxin 4-(1-273). Although insulin stimulated [gamma-(32)P]GTP binding to Rab4 in a time-dependent fashion, its effect on the Rab4 interaction with syntaxin 4 was apparently biphasic; an initial increase in Rab4 associated with syntaxin 4 was followed by a gradual dissociation of the GTPase from syntaxin 4. Finally, the binding of Rab4(Q67L) to GST-syntaxin 4-(1-273) was inhibited by munc-18c in a dose-dependent manner, indicating that GTP-loaded Rab4 binds to syntaxin 4 in the open conformation. These results suggest that 1) Rab4 interacts with syntaxin 4 in a direct and specific manner, and 2) the interaction is regulated by the guanine nucleotide status of Rab4 as well as by the conformational status of syntaxin 4.     

Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound Rab4 and Rab5.

Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiled-coil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rab-binding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTP-bound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.     

Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13.

SNAREs and Rab GTPases cooperate in vesicle transport through a mechanism yet poorly understood. We now demonstrate that the Rab5 effectors EEA1 and Rabaptin-5/Rabex-5 exist on the membrane in high molecular weight oligomers, which also contain NSF. Oligomeric assembly is modulated by the ATPase activity of NSF. Syntaxin 13, the t-SNARE required for endosome fusion, is transiently incorporated into the large oligomers via direct interactions with EEA1. This interaction is required to drive fusion, since both dominant-negative EEA1 and synthetic peptides encoding the FYVE Zn2+ finger hinder the interaction and block fusion. We propose a novel mechanism whereby oligomeric EEA1 and NSF mediate the local activation of syntaxin 13 upon membrane tethering and, by analogy with viral fusion proteins, coordinate the assembly of a fusion pore.     

The small GTPases Rab5a, Rab5b and Rab5c are differentially phosphorylated in vitro.

Rab GTPases play a fundamental role in the regulation of membrane traffic. Three different Rab5 isoforms have been reported but no differences in their function in endocytosis have been discovered. As the Rab5 isoforms show a conserved consensus site for Ser/Thr phosphorylation, we investigated whether this site was phosphorylated. Here, we report that the three Rab5 proteins are differentially recognized by different kinases. Rab5a is efficiently phosphorylated by extracellular-regulated kinase 1 but not by extracellular-regulated kinase 2, while cdc2 kinase preferentially phosphorylates Ser-123 of Rab5b. These findings strongly suggest that phosphorylation could be important to differentially regulate the function of the Rab5 isoforms.     

Determinants of Rab5 interaction with the N terminus of early endosome antigen 1

The Rab5 effector early endosome antigen 1 (EEA1) is a parallel coiled coil homodimer with an N-terminal C(2)H(2) Zn(2+) finger and a C-terminal FYVE domain. Rab5 binds to independent sites at the N and C terminus of EEA1. To gain further insight into the structural determinants for endosome tethering and fusion, we have characterized the interaction of Rab5C with truncation and site-specific mutants of EEA1 using quantitative binding measurements. The results demonstrate that the C(2)H(2) Zn(2+) finger is both essential and sufficient for the N-terminal interaction with Rab5. Although the heptad repeat C-terminal to the C(2)H(2) Zn(2+) finger provides the driving force for stable homodimerization, it does not influence either the affinity or stoichiometry of Rab5 binding. Hydrophobic residues predicted to cluster on a common face of the C(2)H(2) Zn(2+) finger play a critical role in the interaction with Rab5. Although the homologous C(2)H(2) Zn(2+) finger of the Rab5 effector Rabenosyn binds to Rab5 with comparable affinity, the analogous C(2)H(2) Zn(2+) finger of the yeast homologue Vac1 shows no detectable interaction with Rab5, reflecting non-conservative substitutions of critical residues. Large changes in the intrinsic tryptophan fluorescence of Rab5 accompany binding to the C(2)H(2) Zn(2+) finger of EEA1. These observations can be explained by a mode of interaction in which a partially exposed tryptophan residue located at the interface between the switch I and II regions of Rab5 lies within a hydrophobic interface with a cluster of non-polar residues in the C(2)H(2) Zn(2+) finger of EEA1.     

Caveolin-1 activates Rab5 and enhances endocytosis through direct interaction

Caveolin-1, a constitutive protein of the caveolae, is implicated in processes of vesicular transport during caveolae-mediated endocytosis. However, the molecular mechanisms of caveolae-mediated endocytosis are not yet clearly defined. Here, we show the physiological role of the Rab5-caveolin-1 interaction during caveolae-mediated endocytosis. Rab5 was found in caveolae-enriched fractions and Rab5 directly bound to caveolin-1. Furthermore, binding sites of Rab5 to caveolin-1 were identified in the scaffold (SD), transmembrane (TM), and C-terminus (CC) domains, and the Rab5 binding domain of caveolin-1 was required for CTXB uptake. Subsequently, we performed a GST-R5BD pull-down assay to determine whether the Rab5 binding domain of caveolin-1 is involved in Rab5 activity or not. The results showed that overexpression of the Rab5 binding domain of caveolin-1 increase the amount of Rab5-GTP in Cos-1 cells. These findings imply that caveolin-1 controls the Rab5 activity during the caveolae-mediated endocytosis.     

Structure of the APPL1 BAR-PH domain and characterization of its interaction with Rab5

APPL1 is an effector of the small GTPase Rab5. Together, they mediate a signal transduction pathway initiated by ligand binding to cell surface receptors. Interaction with Rab5 is confined to the amino (N)-terminal region of APPL1. We report the crystal structures of human APPL1 N-terminal BAR-PH domain motif. The BAR and PH domains, together with a novel linker helix, form an integrated, crescent-shaped, symmetrical dimer. This BAR-PH interaction is likely conserved in the class of BAR-PH containing proteins. Biochemical analyses indicate two independent Rab-binding sites located at the opposite ends of the dimer, where the PH domain directly interacts with Rab5 and Rab21. Besides structurally supporting the PH domain, the BAR domain also contributes to Rab binding through a small surface region in the vicinity of the PH domain. In stark contrast to the helix-dominated, Rab-binding domains previously reported, APPL1 PH domain employs beta-strands to interact with Rab5. On the Rab5 side, both switch regions are involved in the interaction. Thus we identified a new binding mode between PH domains and small GTPases.     

Direct interaction between Rab3b and the polymeric immunoglobulin receptor controls ligand-stimulated transcytosis in epithelial cells

We have examined the role of rab3b in epithelial cells. In MDCK cells, rab3b localizes to vesicular structures containing the polymeric immunoglobulin receptor (pIgR) and located subjacent to the apical surface. We found that GTP-bound rab3b directly interacts with the cytoplasmic domain of pIgR. Binding of dIgA to pIgR causes a dissociation of the interaction with rab3b, a process that requires dIgA-mediated signaling, Arg657 in the cytoplasmic domain of pIgR, and possibly GTP hydrolysis by rab3b. Binding of dIgA to pIgR at the basolateral surface stimulates subsequent transcytosis to the apical surface. Overexpression of GTP-locked rab3b inhibits dIgA-stimulated transcytosis. Together, our data demonstrate that a rab protein can bind directly to a specific cargo protein and thereby control its trafficking.     

Structural basis of Rab5-Rabaptin5 interaction in endocytosis

Rab5 is a small GTPase that regulates early endosome fusion. We present here the crystal structure of the Rab5 GTPase domain in complex with a GTP analog and the C-terminal domain of effector Rabaptin5. The proteins form a dyad-symmetric Rab5-Rabaptin5(2)-Rab5 ternary complex with a parallel coiled-coil Rabaptin5 homodimer in the middle. Two Rab5 molecules bind independently to the Rabaptin5 dimer using their switch and interswitch regions. The binding does not involve the Rab complementarity-determining regions. We also present the crystal structures of two distinct forms of GDP-Rab5 complexes, both of which are incompatible with Rabaptin5 binding. One has a dislocated and disordered switch I but a virtually intact switch II, whereas the other has its beta-sheet and both switch regions reorganized. Biochemical and functional analyses show that the crystallographically observed Rab5-Rabaptin5 complex also exists in solution, and disruption of this complex by mutation abrogates endosome fusion.     

Rab5 association with the angiotensin II type 1A receptor promotes Rab5 GTP binding and vesicular fusion.

Previous studies have demonstrated that the internalization of the angiotensin II type 1A receptor (AT(1A)R) may be mediated by both beta-arrestin-sensitive and -insensitive mechanisms. Therefore, we have used the AT(1A)R carboxyl-terminal tail to screen a rat brain yeast two-hybrid expression library for novel AT(1A)R-interacting proteins that might contribute to the regulation of AT(1A)R internalization. We have identified Rab5a as an AT(1A)R-binding protein that selectively associates with the AT(1A)R and not with the beta2-adrenergic receptor. A Rab5a-S34N mutant defective in GTP binding does not prevent the internalization of the AT(1A)R but does prevent the trafficking of the AT(1A)R into larger hollow cored vesicular structures. Agonist activation of the AT(1A)R promotes both the formation of Rab5a.AT(1A)R protein complexes and Rab5a GTP binding. Rab5a interactions with the AT(1A)R are mediated in part by the last 10 amino acid residues of the AT(1A)R carboxyl-terminal tail, and although a mutant receptor lacking these residues internalizes normally, it does not redistribute into larger hollow vesicles. Our data suggest that AT(1A)R activation modulates Rab5a activity leading to the homotypic fusion of endocytic vesicles. These observations suggest that vesicular cargo proteins, such as the AT(1A)R, may control their targeting between intracellular compartments by directly regulating the activity of components of the intracellular trafficking machinery such as Rab5a.     

Direct interaction with Rab11a targets the epithelial Ca2+ channels TRPV5 and TRPV6 to the plasma membrane

TRPV5 and TRPV6 are the most Ca2+-selective members of the transient receptor potential (TRP) family of cation channels and play a pivotal role in the maintenance of Ca2+ balance in the body. However, little is known about the mechanisms controlling the plasma membrane abundance of these channels to regulate epithelial Ca2+ transport. In this study, we demonstrated the direct and specific interaction of GDP-bound Rab11a with TRPV5 and TRPV6. Rab11a colocalized with TRPV5 and TRPV6 in vesicular structures underlying the apical plasma membrane of Ca2+-transporting epithelial cells. This GTPase recognized a conserved stretch in the carboxyl terminus of TRPV5 that is essential for channel trafficking. Furthermore, coexpression of GDP-locked Rab11a with TRPV5 or TRPV6 resulted in significantly decreased Ca2+ uptake, caused by diminished channel cell surface expression. Together, our data demonstrated the important role of Rab11a in the trafficking of TRPV5 and TRPV6. Rab11a exerts this function in a novel fashion, since it operates via direct cargo interaction while in the GDP-bound configuration.     

Direct interaction of the Rab3 effector RIM with Ca2+ channels, SNAP-25, and synaptotagmin.

To define the role of the Rab3-interacting molecule RIM in exocytosis we searched for additional binding partners of the protein. We found that the two C(2) domains of RIM display properties analogous to those of the C(2)B domain of synaptotagmin-I. Thus, RIM-C(2)A and RIM-C(2)B bind in a Ca(2+)-independent manner to alpha1B, the pore-forming subunit of N-type Ca(2+) channels (EC(50) = approximately 20 nm). They also weakly interact with the alpha1C but not the alpha1D subunit of L-type Ca(2+) channels. In addition, the C(2) domains of RIM associate with SNAP-25 and synaptotagmin-I. The binding affinities for these two proteins are 203 and 24 nm, respectively, for RIM-C(2)A and 224 and 16 nm for RIM-C(2)B. The interactions of the C(2) domains of RIM with SNAP-25 and synaptotagmin-I are modulated by Ca(2+). Thus, in the presence of Ca(2+) (EC(50) = approximately 75 microm) the interaction with synaptotagmin-I is increased, whereas SNAP-25 binding is reduced. Synaptotagmin-I binding is abolished by mutations in two positively charged amino acids in the C(2) domains of RIM and by the addition of inositol polyphosphates. We propose that the Rab3 effector RIM is a scaffold protein that participates through its multiple binding partners in the docking and fusion of secretory vesicles at the release sites.     

Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF.

Insulin-like growth factor (IGF)-1 plays crucial roles in growth control and rearrangements of the cytoskeleton. IGF-1 binds to the IGF-1 receptor and thereby induces the autophosphorylation of this receptor at its tyrosine residues. The phosphorylation of the IGF-1 receptor is thought to initiate a cascade of events. Although various signaling molecules have been identified, they appear to interact with the tyrosine-phosphorylated IGF-1 receptor. Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule. LARG formed a complex with the IGF-1 receptor in vivo, and the PDZ domain of LARG interacted directly with the COOH-terminal domain of IGF-1 receptor in vitro. LARG had an exchange activity for Rho in vitro and induced the formation of stress fibers in NIH 3T3 fibroblasts. When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced. Furthermore, the IGF-1-induced Rho-kinase activation and the enhancement of stress fibers were inhibited by ectopic expression of the PDZ and RGS domains of LARG. Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.     

Multivalent endosome targeting by homodimeric EEA1.

Early endosome autoantigen localization to early endosomes is mediated by a C-terminal region, which includes a calmodulin binding motif, a Rab5 interaction site, and a FYVE domain that selectively binds phosphatidyl inositol 3-phosphate. The crystal structure of the C-terminal region bound to inositol 1,3-bisphosphate reveals an organized, quaternary assembly consisting of a parallel coiled coil and a dyad-symmetric FYVE domain homodimer. Structural and biochemical observations support a multivalent mechanism for endosomal localization in which domain organization, dimerization, and quaternary structure amplify the weak affinity and modest specificity of head group interactions with conserved residues. A unique mode of membrane engagement deduced from the quaternary structure of the C-terminal region provides insight into the structural basis of endosome tethering.