Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology.

Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.

Borg proteins control septin organization and are negatively regulated by Cdc42.

The Cdc42 GTPase binds to numerous effector proteins that control cell polarity, cytoskeletal remodelling and vesicle transport. In many cases the signalling pathways downstream of these effectors are not known. Here we show that the Cdc42 effectors Borg1 to Borg3 bind to septin GTPases. Endogenous septin Cdc10 and Borg3 proteins can be immunoprecipitated together by an anti-Borg3 antibody. The ectopic expression of Borgs disrupts normal septin organization. Cdc42 negatively regulates this effect and inhibits the binding of Borg3 to septins. Borgs are therefore the first known regulators of mammalian septin organization and provide an unexpected link between the septin and Cdc42 GTPases.

Rabphilin dissociated from Rab3 promotes endocytosis through interaction with Rabaptin-5.

Rabphilin is a secretory vesicle protein that interacts with the GTP-bound form of the small GTPase Rab3. We investigated the involvement of Rabphilin in endocytosis using different point mutants of the protein. Overexpression of wild-type Rabphilin in the insulin-secreting cell line HIT-T15 did not affect receptor-mediated transferrin endocytosis. By contrast, Rabphilin V61A, a mutant that is unable to interact with Rab3, enhanced the rate of transferrin internalization. The effect of Rabphilin V61A was not mimicked by Rabphilin L83A, another mutant with impaired Rab3 binding. Careful analysis of the properties of the two mutants revealed that Rabphilin V61A and Rabphilin L83A are both targeted to secretory vesicles, have stimulatory activity on exocytosis, and bind equally well to alpha-actinin. However, Rabphilin L83A fails to interact with Rabaptin-5, an important component of the endocytotic machinery. These results indicate that Rabphilin promotes receptor-mediated endocytosis and that its action is negatively modulated by Rab3. We propose that the hydrolysis of GTP that is coupled to the exocytotic event disrupts the Rabphilin-Rab3 complex and permits the recruitment of Rabaptin-5 at the fusion site. Our data show that immediately after internalization the transferrin receptor and VAMP-2 colocalize on the same vesicular structures, suggesting that Rabphilin favors the rapid recycling of the components of the secretory vesicle.

PIST: a novel PDZ/coiled-coil domain binding partner for the rho-family GTPase TC10.

TC10 is a member of the Rho family of GTPases, most closely related to Cdc42. This family of proteins mediates cytoskeletal rearrangements, activation of signal transduction cascades, and activation of gene transcription. A current focus is to identify and characterize the GTPase effectors that are involved in these cellular events. Many specific effectors for Cdc42 have been identified, most of which bind equally well to TC10, though a subset has only a low affinity for TC10. No protein that specifically interacts with TC10 has yet been described. Here, we report the cloning and characterization of PIST, a TC10-specific interacting protein. PIST possesses a PDZ domain and two, putative, coiled-coil domains, one of which contains a leucine zipper. It interacts directly and specifically with TC10:GTP, though with low affinity, and a mutation within the effector binding domain of TC10 disrupts the interaction. PIST also forms homodimers. The first coiled-coil and PDZ domains are not necessary for these interactions, but deletion of the N-terminal portion of the leucine zipper abolishes dimerization. PIST may function as a scaffolding protein to link TC10 to signaling pathways.

The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42.

PAR (partitioning-defective) proteins, which were first identified in the nematode Caenorhabditis elegans, are essential for asymmetric cell division and polarized growth, whereas Cdc42 mediates establishment of cell polarity. Here we describe an unexpected link between these two systems. We have identified a family of mammalian Par6 proteins that are similar to the C. elegans PDZ-domain protein PAR-6. Par6 forms a complex with Cdc42-GTP, with a human homologue of the multi-PDZ protein PAR-3 and with the regulatory domains of atypical protein kinase C (PKC) proteins. This assembly is implicated in the formation of normal tight junctions at epithelial cell-cell contacts. Thus, Par6 is a key adaptor that links Cdc42 and atypical PKCs to Par3.

High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion.

Rabphilin is a protein that associates with the GTP-bound form of Rab3, a small GTPase that controls a late step in Ca(2+)-triggered exocytosis. Rabphilin is found only in neuroendocrine cells where it co-localises with Rab3A on the secretory vesicle membrane. The Rab3 binding domain (residues 45 to 170), located in the N-terminal part of Rabphilin, includes a cysteine-rich region with two zinc finger motifs that are required for efficient interaction with the small GTPase. To determine whether binding to Rab3A is necessary for the subcellular localisation of Rabphilin, we synthesised point mutants within the Rab3-binding domain. We found that two unique mutations (V61A and L83A) within an amphipathic alpha-helix of this region abolish detectable binding to endogenous Rab3, but only partially impair the targetting of the protein to secretory vesicles in PC12 and pancreatic HIT-T15 cells. Furthermore, both mutants transfected in the HIT-T15 beta cell line stimulate Ca(2+)-regulated exocytosis to the same extent as wild-type Rabphilin. Surprisingly, another Rabphilin mutant, R60A, which possesses a wild-type affinity for Rab3, and targets efficiently to membranes, does not potentiate regulated secretion. High affinity binding to Rab3 is therefore dispensable for the targetting of Rabphilin to secretory vesicles and for the potentiation of Ca(2+)-regulated secretion. The effects of Rabphilin on secretion may be mediated through interaction with another, unknown, factor that recognizes the Rab3 binding domain.

The Borgs, a new family of Cdc42 and TC10 GTPase-interacting proteins.

The Rho family of GTPases plays key roles in the regulation of cell motility and morphogenesis. They also regulate protein kinase cascades, gene expression, and cell cycle progression. This multiplicity of roles requires that the Rho GTPases interact with a wide variety of downstream effector proteins. An understanding of their functions at a molecular level therefore requires the identification of the entire set of such effectors. Towards this end, we performed a two-hybrid screen using the TC10 GTPase as bait and identified a family of putative effector proteins related to MSE55, a murine stromal and epithelial cell protein of 55 kDa. We have named this family the Borg (binder of Rho GTPases) proteins. Complete open reading frames have been obtained for Borg1 through Borg3. We renamed MSE55 as Borg5. Borg1, Borg2, Borg4, and Borg5 bind both TC10 and Cdc42 in a GTP-dependent manner. Surprisingly, Borg3 bound only to Cdc42. An intact CRIB (Cdc42, Rac interactive binding) domain was required for binding. No interaction of the Borgs with Rac1 or RhoA was detectable. Three-hemagglutinin epitope (HA(3))-tagged Borg3 protein was mostly cytosolic when expressed ectopically in NIH 3T3 cells, with some accumulation in membrane ruffles. The phenotype induced by Borg3 was reminiscent of that caused by an inhibition of Rho function and was reversed by overexpression of Rho. Surprisingly, it was independent of the ability to bind Cdc42. Borg3 also inhibited Jun kinase activity by a mechanism that was independent of Cdc42 binding. HA(3)-Borg3 expression caused substantial delays in the spreading of cells on fibronectin surfaces after replating, and the spread cells lacked stress fibers. We propose that the Borg proteins function as negative regulators of Rho GTPase signaling.

Temporal association of the N- and O-linked glycosylation events and their implication in the polarized sorting of intestinal brush border sucrase-isomaltase, aminopeptidase N, and dipeptidyl peptidase IV.

The temporal association between O-glycosylation and processing of N-linked glycans in the Golgi apparatus as well as the implication of these events in the polarized sorting of three brush border proteins has been the subject of the current investigation. O-Glycosylation of pro-sucrase-isomaltase (pro-SI), aminopeptidase N (ApN), and dipeptidyl peptidase IV (DPPIV) is drastically reduced when processing of the mannose-rich N-linked glycans is blocked by deoxymannojirimycin, an inhibitor of the Golgi-located mannosidase I. By contrast, O-glycosylation is not affected in the presence of swainsonine, an inhibitor of Golgi mannosidase II. The results indicate that removal of the outermost mannose residues by mannosidase I from the mannose-rich N-linked glycans is required before O-glycosylation can ensue. On the other hand, subsequent mannose residues in the core chain impose no sterical constraints on the progression of O-glycosylation. Reduction or modification of N- and O-glycosylation do not affect the transport of pro-SI, ApN, or DPPIV to the cell surface per se. However, the polarized sorting of two of these proteins, pro-SI and DPPIV, to the apical membrane is substantially altered when O-glycans are not completely processed, while the sorting of ApN is not affected. The processing of N-linked glycans, on the other hand, has no influence on sorting of all three proteins. The results indicate that O-linked carbohydrates are at least a part of the sorting mechanism of pro-SI and DPPIV. The sorting of ApN implicates neither O-linked nor N-linked glycans and is driven most likely by carbohydrate-independent mechanisms.

Comparison of the effects on secretion in chromaffin and PC12 cells of Rab3 family members and mutants. Evidence that inhibitory effects are independent of direct interaction with Rabphilin3.

The Rab class of low molecular weight GTPases has been implicated in the regulation of vesicular trafficking between membrane compartments in eukaryotic cells. The Rab3 family consisting of four highly homologous isoforms is associated with secretory granules and synaptic vesicles. Many different types of experiments indicate that Rab3a is a negative regulator of exocytosis and that its GTP-bound form interacts with Rabphilin3, a possible effector. Overexpression of Rabphilin3 in chromaffin cells enhances secretion. We have investigated the expression, localization, and effects on secretion of the various members of the Rab3 family in bovine chromaffin and PC12 cells. We found that Rab3a, Rab3b, Rab3c, and Rab3d are expressed to varying degrees in PC12 cells and in a fraction enriched in chromaffin granule membranes from the adrenal medulla. Immunocytochemistry revealed that all members of the family when overexpressed in PC12 cells localize to secretory granules. Binding constants for the interaction of the GTP-bound forms of Rab3a, Rab3b, Rab3c, and Rab3d with Rabphilin3 were comparable (Kd = 10-20 nM). Overexpression of each of the four members of the Rab3 family inhibited secretion. Mutations in Rab3a were identified that strongly impaired the ability of the GTP-bound form to interact with Rabphilin3. The mutated proteins inhibited secretion similarly to wild type Rab3a. Although Rab3a and Rabphilin3 are located on the same secretory granule or secretory vesicle and interact both in vitro and in situ, it is concluded that the inhibition of secretion by overexpression of Rab3a is unrelated to its ability to interact with Rabphilin3.

Distinct cellular effects and interactions of the Rho-family GTPase TC10.

BACKGROUND: Rho-family GTPases have central roles in cytoskeletal organization, proliferation, differentiation and apoptosis. Multiple factors possessing overlapping specificities for Rho GTPases have been identified. The Rho GTPases Cdc42 and Rac share many regulators and effectors, yet produce different phenotypes when expressed as gain-of-function mutants in cells. The Rho-family member TC10 has remained almost completely uncharacterized, so it was of interest to determine whether TC10 has unique cellular effects and interacts with the same targets as Cdc42 and Rac. RESULTS: A gain-of-function TC10 mutant protein expressed in fibroblasts induced cell rounding, loss of stress fibers and formation of peripheral extensions. The extensions were longer than those induced by the analogous Cdc42 mutant protein. Cells expressing TC10 also possessed fewer membrane ruffles and stress fibers than those expressing Cdc42. TC10 mRNA was most highly expressed in heart and skeletal muscle. The GTPase activity of TC10 was lower than that of Cdc42, and TC10 possessed a lower affinity for, but greater responsiveness to, the p50Rho GTPase-activating protein (p50RhoGAP) than did Cdc42. TC10 stimulated Jun N-terminal kinase (JNK) and p21-activated kinase (PAK) activities and interacted with a set of effectors (alpha-, beta- and gammaPAK, MRCKalpha/beta, MLK2, N-WASP and MSE55) that overlaps with those for Cdc42 and Rac. TC10 did not interact with MLK3 or WASP, and interacted only weakly with ACK-1. CONCLUSIONS: TC10 possesses distinct features, but exhibits a phenotype most closely related to that of Cdc42. It interacts with a similar subset of effectors to Cdc42 but not with MLK3, WASP or ACK-1. It is regulated differentially by p50RhoGAP.

Protein domains implicated in intracellular transport and sorting of lactase-phlorizin hydrolase.

The roles of various domains of intestinal lactase-phlorizin hydrolase (pro-LPH) on its folding, dimerization, and polarized sorting are investigated in deletion mutants of the ectodomain fused or not fused with the membrane-anchoring and cytoplasmic domains (MACT). Deletion of 236 amino acids immediately upstream of MACT has no effect on the folding, dimerization, transport competence, or polarized sorting of the mutant LPH1646MACT. By contrast, LPH1646, an anchorless counterpart of LPH1646MACT, is not transported beyond the ER and persists as a mannose-rich monomer during its entire life cycle. The further deletion of 87 amino acids generates a correctly folded but transport-incompetent monomeric LPH1559MACT mutant. The results strongly suggest that dimerization and transport of pro-LPH implicate a stretch of 87 amino acids in the ectodomain between LPH1646MACT and LPH1559MACT. In addition, dimerization of pro-LPH requires at least two further criteria: (i) a correctly folded ectodomain of pro-LPH and (ii) the presence of the transmembrane region. Neither of these requirements alone is sufficient for dimerization. Finally, the sorting of pro-LPH appears to be mediated by signals located between the cleavage site of pro-LPH and the LPH1646MACT mutant.

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

Isoprenylation of Rab proteins possessing a C-terminal CaaX motif.

Rab proteins are small GTPases highly related to the yeast Ypt1 and Sec4 proteins involved in secretion. The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways. They share distinct C-terminal cysteine motifs required for membrane association. Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C-terminal CaaX motif similar to those of Ras/Rho proteins. This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro. Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate. Our study shows that the CaaX motif is required for isoprenylation.