Rab Isoforms Research Articles

Large Scale Screening for Novel Rab Effectors Reveals Unexpected Broad Rab Binding Specificity

Small GTPase Rab is generally thought to control intracellular membrane trafficking through interaction with specific effector molecules. Because of the large number of Rab isoforms in mammals, however, the effectors of most of the mammalian Rabs have never been identified, and the Rab binding specificity of the Rab effectors previously reported has never been thoroughly investigated. In this study we systematically screened for novel Rab effectors by a yeast two-hybrid assay with 28 different mouse or human Rabs (Rab1-30) as bait and identified 27 Rab-binding proteins, including 19 novel ones. We further investigated their Rab binding specificity by a yeast two-hybrid assay with a panel of 60 different GTP-locked mouse or human Rabs. Unexpectedly most (17 of 27) of the Rab-binding proteins we identified exhibited broad Rab binding specificity and bound multiple Rab isoforms. As an example, inositol-polyphosphate 5-phosphatase OCRL (oculocerebrorenal syndrome of Lowe) bound the greatest number of Rabs (i.e. 16 distinct Rabs). Others, however, specifically recognized only a single Rab isoform or only two closely related Rab isoforms. The interaction of eight of the novel Rab-binding proteins identified (e.g. INPP5E and Cog4) with a specific Rab isoform was confirmed by co-immunoprecipitation assay and/or colocalization analysis in mammalian cell cultures, and the novel Rab2B-binding domain of Golgi-associated Rab2B interactor (GARI) and GARI-like proteins was identified by deletion and homology search analyses. The findings suggest that most Rab effectors (or Rab-binding proteins) regulate intracellular membrane trafficking through interaction with several Rab isoforms rather than through a single Rab isoform.

Regulation of ENaC by small G proteins

Small G proteins are recognized to regulate diverse cellular processes. We have shown previously that the epithelial Na+ channel (ENaC) is regulated by Ras and Rho GTPases. K‐Ras, RhoA and Rac1 markedly increase activity of ENaC reconstituted in CHO cells in a GTP‐dependent manner. However, their transductionpathways and mechanisms of activation are different; K‐Ras increases channel open probability via PI3‐kinase signaling, whereas RhoA increase channel membrane levels. Regulation of ENaC open probability by PI3‐kinaseis a direct consequence of the physical association of the product of PI3‐kinase, PI(3,4,5)P3with the channel. RhoA activates ENaC via Rho‐kinase and subsequent activation of PI(4)P5‐kinase with concomitant increases in PI(4,5)P2 levels promoting channel insertion into the plasma membrane. Recent studies reveal that multiple Rab isoforms physically interact with and/or modulate the activity of several ion channels. Rab11a is well‐established as a participant in the regulation of recycling endosomal trafficking. Rab11a is associated with vesicles in the apical portion of epithelial cells near the centrosome and beneath the apical plasma membrane. Co‐expression of Rab11a with ENaC results in a significant increase in channel activity. We conclude that Rab11a may be important in the trafficking of ENaC.Supported by AHA.

Regulation of epithelial ion channels by Rab GTPases

Epithelial ion channels are crucial to many of life’s processes and disruption of their functions can lead to several disorders. Cystic fibrosis, an autosomal recessive disorder, is caused by defects in the biosynthesis or function of the CFTR chloride channel. Similarly, mutations in certain ENaC genes leading to increased or reduced channel activity cause diseases such as Liddle’s syndrome or PHA. In order for ion channel proteins to be functional they need to be expressed on the plasma membrane. Thus, molecules that modulate the trafficking of ion channels to and from the membrane are of utmost significance. Among the numerous factors that regulate their functioning is a family of small GTPases known as Rab proteins. While Rabs have always played a pivotal role in membrane trafficking, their diversity of functions and plethora of interacting partners have lately been brought to light. Recent studies reveal that multiple Rab isoforms physically interact with and/or modulate the activity of several ion channels. Rab proteins have the ability to serve as molecular switches and many of the ion channels are regulated differentially by the GTP- or GDP-bound Rab isoforms. This review examines the role of Rab GTPases in the trafficking of ion channels, including CFTR, ENaC, TRPV5/6, and aquaporins, based on recent evidence.

Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells

ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein–protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension.

Rab and SNARE isoform expression in cytotoxic T-cells (CTL): Potential players in regulated release of granzyme-B (GrB)

Rationale Cytotoxic T-cells (CTLs, CD8 + lymphocytes) have been implicated in asthma and chronic obstructive pulmonary disease (COPD), however the mechanism of CTL cytotoxic granule secretion of granzyme-B (GrB) is uncertain. Secretory cell exocytosis is dependent on interactions between SNARE (soluble nSF attachment protein receptor) proteins on vesicular (v-SNAREs) and target plasma membrane (t-SNARE). Rab proteins regulate intracellular organelle transport, matching appropriate v- with t-SNAREs. We hypothesized that SNARE and Rab proteins are involved in the regulated release of GrB stored in CTLs and natural killer (NK) cells. Methods Specific primer sets determined SNARE and Rab mRNA expression in human CD8 + cells and YT-Indy, a human NK cell line, by RT-PCR. Western blot analysis and immunocytochemistry, using specific antibodies, confirmed SNARE and Rab protein expression. Results CTLs and YT-Indy were shown to express mRNA for the Rab proteins, Rab 4a, 5a, and 27a. CTLs encoded for the v-SNAREs, VAMP 1, 2, 8, and the t-SNAREs, syntaxin 3, 4, 6, and SNAP-23. YT-Indy cells expressed mRNA for the SNAREs, VAMP 2, 7, 8, syntaxin-3, and SNAP-23. Expression of syntaxin-4, and 6 in the YT-Indy cell line was, however, variable. Interestingly, mRNA for VAMP-1 was expressed in CTLs, but not in YT-Indy, which may suggest cell-specific differences in regulation of secretion. Protein expression for SNAP-23, syntaxin-4, syntaxin-6, and VAMP-8 on GrB + granule membranes in CTLs was detected by Western blot analysis. Conclusions Our data suggest that SNARE proteins and Rabs may be important in the regulation of CTL mediated release of GrB.

Slp4-a/Granuphilin-a Regulates Dense-core Vesicle Exocytosis in PC12 Cells

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a was originally identified as a protein specifically associated with insulin-containing vesicles in pancreatic beta-cells (Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999) J. Biol. Chem. 274, 28542-28548). Previously, we showed that the N-terminal Slp homology domain of Slp4-a interacts with the GTP-bound form of Rab3A, Rab8, and Rab27A both in vitro and in intact cells (Kuroda, T. S., Fukuda, M., Ariga, H., and Mikoshiba, K. (2002) J. Biol. Chem. 277, 9212-9218). How Slp4-a.Rab complex controls regulated secretion, and which Rab isoforms dominantly interact with Slp4-a in vivo, however, have remained unknown. In this study, we showed by immunocytochemistry and subcellular fractionation that three Rabs, Rab3A, Rab8, and Rab27A, and Slp4-a are endogenously expressed in neuroendocrine PC12 cells and localized on dense-core vesicles, and we discovered that the Slp4-a.Rab8 and Slp4-a.Rab27A complexes, but not Slp4-a.Rab3A complexes, are formed on dense-core vesicles in PC12 cells, although the majority of Rab8 is present in the cell body and is free of Slp4-a. We further showed that expression of Rab27A, but not of Rab8, promotes high KCl-dependent secretion of neuropeptide Y (NPY) in PC12 cells, whereas expression of Slp4-a, but not of an Slp4-a mutant incapable of Rab27A binding, inhibits NPY secretion in PC12 cells. In contrast, expression of Slp3-a, but not of Slp3-b lacking an N-terminal Rab27A-binding domain, promotes NPY secretion. These findings suggest that the Slp family controls regulated dense-core vesicle exocytosis via binding to Rab27A.

A study on CD45 isoform expression during T-cell development and selection events in the human thymus

CD45 molecules are known to appear as various isoforms generated by alternative splicing of variable exons 4, 5, and 6, but the detailed profile of CD45 isoform expression during thymocyte development has not been revealed. We examined the CD45 isoforms expressed in the various human thymocytes’ subsets defined by CD3, CD4, and CD8 expressions using RT-PCR and 4-color flow cytometry. RT-PCR study revealed that RABC, RAB, RBC, RB, and R0 isoforms were expressed in thymocytes while any of RAC, RA, or RC isoforms were not detected. RABC, RAB and RBC isoforms were expressed at CD3 −CD4 −CD8 − and CD3 +CD4 +CD8 − stages, but were barely detectable at CD3 −CD4 +CD8 + stage. RB isoform was consistently expressed at a relatively high level at all stages. R0 isoform was expressed at a low level at CD3 −CD4 −CD8 − and CD3 −CD4 +CD8 − stages but upregulated at CD3 +CD4 +CD8 + and CD3 +CD4 +CD8 − stages. In combination with the results obtained by 4-color flow cytometric study, CD45 isoform expression on human thymocytes were determined to be RABC +RAB ±RBC +RB +R0 ± at CD3 −CD4 −CD8 − stage, RABC −RAB −RBC −RB +R0 + at CD3 −CD4 +CD8 − and CD3 −CD4 +CD8 + stages, RABC ±RAB +RBC +RB ++R0 ++ at CD3 +CD4 +CD8 + stage, and RABC +RAB +RBC +RB ++R0 + at CD3 +CD4 +CD8 − stage. Bcl-2 expression was upregulated between CD3 −CD4 +CD8 +CD45R0 + and CD3 +CD4 +CD8 +CD45R0 + stages. Expression of CD45R0 epitope was upregulated between CD3 −CD4 +CD8 +CD69 − and CD3 +CD4 +CD8 +CD69 + stages while CD45RA epitope expression was unchanged. Thus, when thymocytes are positively selected, CD45R0 isoform expression seems to be upregulated while CD45RABC isoform expression stays at a very low level. In summary, various isoforms of CD45 were shown to be tightly regulated during thymocyte development and through the selection process.

Interferon-γ Selectively Induces Rab5a Synthesis and Processing in Mononuclear Cells

Macrophage activation by interferon (IFN)-gamma is characterized by enhanced phagocytosis and killing of internalized pathogens. We studied the effects of IFN-gamma on Rab5a, a GTPase involved in both endocytosis and phagocytosis. IFN-gamma induced the synthesis of Rab5a in mononuclear cells as detected by immunoprecipitation and by Western blotting. Rab5a messenger RNA levels were also increased. Elevated protein expression was detected as early as 6 h following IFN-gamma and was maximal at 24 h. Following IFN-gamma, membrane association of Rab5a:GTP was substantially increased. Rab5b and Rab5c, as well as Rab7 and Rab11, Rab GTPases localized in the endosomal-lysosomal pathway, were unaffected by IFN-gamma. Moreover, Rab5a expression in non-macrophages was unaltered by IFN-gamma. Rab5a is a prenylated protein, and newly synthesized Rab5a was rapidly processed following IFN-gamma. However, elevated geranylgeranylation was not Rab5a-specific since all the Rab5 isoforms were more rapidly prenylated in vitro using cytosol from IFN-gamma-treated cells. Last, guanine nucleotide exchange on Rab5a was elevated about 3-fold in the presence of cytosol from IFN-gamma-treated cells. The selective effect of IFN-gamma on Rab5a, synthesis, processing, and nucleotide exchange suggests that Rab isoforms have closely associated but not identical functions and that selective enhancement of membrane trafficking may play a key role in intracellular killing.

Cloning of Rab GTPases expressed in human skeletal muscle: studies in insulin-resistant subjects.

To explore the potential role of Rab GTPases in human insulin resistance, we first employed a PCR-cloning approach to identify Rab isoforms that are expressed in human skeletal muscle. Multiple Rab isoforms including Rab1A, Rab4A, Rab5B, Rab7, Rab8, Rab10, Rab12A, Rab13, Rab18, Rab21, and Rab22 mRNA were found to be expressed in human skeletal muscle. The second goal was to examine whether mRNA expression for Rabs targeted to endocytotic/exocytotic compartments was altered as a function of insulin resistance. Quantitative PCR analysis demonstrated that Rab4A, Rab5B and Rab18 mRNA levels in skeletal muscle from insulin-resistant patients without (IR) and with non-insulin-dependent diabetes mellitus (NIDDM) were not significantly different from those in insulin-sensitive controls (IS). At the protein level, total Rab5B amount was not significantly different among IS, IR and NIDDM subgroups. However, in basal muscle, Rab5B in the total membrane fraction was 2.1-3.6 fold higher in IR and NIDDM than in IS subjects. Insulin increased membrane-associated Rab5B by 3-fold in IS subjects, whereas this effect was not significant in both IR and NIDDM subgroups. Thus, for the first time, we have comprehensively studied the mRNA expression of Rab isoforms in human muscle. The phlethora of Rab GTPases are indicative of high volume of vesicular traffic and regulated metabolism. The potential role of specific Rab isoforms in insulin resistance does not rely on a change in steady state mRNA levels, but is demonstrable as an alteration in protein subcellular distribution and trafficking.

Molecular Cloning and Characterization of Rab27a and Rab27b, Novel Human Rab Proteins Shared by Melanocytes and Platelets

Rabs are prenylated, membrane-bound proteins involved in vesicular fusion and trafficking. We isolated the complete cDNAs of two rab isoforms, rab27a and rab27b, from human melanoma cells and melanocytes. Rab27a is the human homolog of a rat megakaryocyte rab called ram p25. Rab27b corresponds to a small GTP-binding protein, c25KG, which was previously purified from platelets but whose cDNA had not been cloned. Sequence comparisons with known rabs indicate that rab27a and rab27b comprise a melanocyte/platelet subfamily within the rab family. In addition, rab27a was expressed in a large variety of cell and tissue types, excluding brain, and rab27b manifested itself primarily in testis. Bacterially expressed and purified rab27a and rab27b exhibited GTP-binding activity and can now be used for antibody production and studies of the substrate specificities of geranylgeranyl transferase. In addition, the expression of rab27a and rab27b in both melanocytes and platelets makes them candidates for involvement in mouse and human disorders characterized by the combination of pigment dilution and a platelet storage pool defect.

Abundance and subcellular distribution of GTP‐binding proteins in 3T3‐L1 cells before and after differentiation to the insulin‐sensitive phenotype

The abundance and the subcellular distribution of GTP-binding proteins was studied in membrane fractions (plasma membranes and low-density microsomes) from 3T3-L1 cells before and after differentiation to the insulin-sensitive phenotype. After differentiation, the abundance of alpha i (alpha subunit of GTP-binding protein Gi), alpha o (alpha subunit of GTP-binding protein G(o)), and of a 47-kDa alpha s (alpha subunit of GTP-binding protein Gs) as detected by immunoblotting with specific antisera was reduced by 10-50% when normalized per membrane protein. In contrast, a 43-kDa alpha s was increased about threefold after differentiation. Furthermore, cholera-toxin-catalyzed ADP-ribosylation of both 43-kDa and 47-kDa alpha s was disproportionately increased ninefold and threefold, respectively, possibly reflecting the increased production of an ADP-ribosylation factor in the differentiated cells. The small GTP-binding protein Ha-ras was reduced by 50%, whereas rab1 and other small GTP-binding proteins tentatively identified as rab-isoforms (ras-homologous gene products from brain) were increased by 100% and 70%, respectively. Since the total protein content of 3T3-L1 cells was increased threefold after differentiation, the observed increase of the 43-kDa alpha s, rab1 and of the other rab isoforms was eightfold, sixfold and fivefold, respectively, when normalized/cell count. With the exception of the rab isoforms, all GTP-binding proteins were predominantly, if not exclusively, located in the plasma membrane; comparable amounts of the rab isoforms were found in plasma membranes and low-density microsomes. Insulin induced the characteristic redistribution of glucose transporters GLUT4 from low-density microsomes to the plasma membranes, but failed to alter the subcellular distribution of any of the GTP-binding proteins investigated. These data suggest that the increase in the abundance of the 43-kDa alpha s subunit and of several rab isoforms might be related to specific functions of the adipocyte-like phenotype, but that none of the investigated guanine-nucleotide-binding regulatory (G)-proteins appears to be tightly associated with the GLUT4.