Rab5-activating protein 6, a novel endosomal protein with a role in endocytosis

Rab GTPases are regulators of membrane trafficking that cycle between active (GTP-bound) and inactive (GDP-bound) states. In this study, we report the identification of a new human Rab5 guanine nucleotide exchange factor (GEF), which we have named RAP6 (Rab5-activating protein 6). RAP6 contains a Rab5 GEF and a Ras GAP domain. We show that the Vps9 domain is sufficient for the interaction of RAP6 with GDP-bound Rab5 and that RAP6 stimulates Rab5 guanine nucleotide exchange. We also find that the Ras GAP domain of RAP6 shows GAP activity for Ras. Immunofluorescence experiments reveal that RAP6 is associated with plasma membrane and small intracellular vesicles that also contain Rab5. Additionally, the overexpression of RAP6 affects both fluid phase and receptor-mediated endocytosis. This study is the first to show that RAP6 is a novel regulator of endocytosis that exhibits GEF activity specific for Rab5 and GAP activity specific for Ras.     

Rin1 regulates insulin receptor signal transduction pathways

Rin1 is a multifunctional protein containing several domains, including Ras binding and Rab5 GEF domains. The role of Rin1 in insulin receptor internalization and signaling was examined by expressing Rin1 and deletion mutants in cells utilizing a retrovirus system. Here, we show that insulin-receptor-mediated endocystosis and fluid phase insulin-stimulated endocytosis are enhanced in cells expressing the Rin1:wild type and the Rin1:C deletion mutant, which contain both the Rab5-GEF and GTP-bound Ras binding domains. However, the Rin1:N deletion mutant, which contains both the SH2 and proline-rich domains, blocked insulin-stimulated receptor-mediated and insulin-stimulated fluid phase endocytosis. In addition, the expression of Rin1:delta (429-490), a natural occurring splice variant, also blocked both receptor-mediated and fluid phase endocystosis. Furthermore, association of the Rin1 SH2 domain with the insulin receptor was dependent on tyrosine phosphorylation of the insulin receptor. Morphological analysis indicates that Rin1 co-localizes with insulin receptor both at the cell surface and in endosomes upon insulin stimulation. Interestingly, the expression of Rin1:wild type and both deletion mutants blocks the activation of Erk1/2 and Akt1 kinase activities without affecting either JN or p38 kinase activities. DNA synthesis and Elk-1 activation are also altered by the expression of Rin1:wild type and the Rin1:C deletion mutant. In contrast, the expression of Rin1:delta stimulates both Erk1/2 and Akt1 activation, DNA synthesis and Elk-1 activation. These results demonstrate that Rin1 plays an important role in both insulin receptor membrane trafficking and signaling.     

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.     

Physical-chemical influences on vernal zooplankton community structure in small lakes and wetlands of Wisconsin,U.S.A.

We sampled zooplankton communities from 54 small water bodies distributed throughout Wisconsin to evaluate whether a snap-shot of zooplankton community structure during early spring could be used for the purpose of differentiating lakes from wetlands. We collected a single set of zooplankton and water chemistry data during a one-month time window (synchronized from south to north across the state) from an open water site in each basin as a means to minimize and standardize sampling effort and to minimize cascading effects arising from predator–prey interactions with resident and immigrant aquatic insect communities. We identified 53 taxa of zooplankton from 54 sites sampled across Wisconsin. There was an average of 6.83 taxa per site. The zooplankton species were distributed with a great deal of independence. We did not detect significant correlations between number of taxa and geographic region or waterbody size. There was a significant inverse correlation between number of taxa and the concentration of calcium ion, alkalinity and conductivity. One pair of taxa, Lynceus brachyurus and Chaoborus americanus, showed a significant difference in average duration of sites of their respective occurrence. All other pairs of taxa had no significant difference in average latitude, waterbody surface area, total phosphorus, total Kjeldahl nitrogen, alkalinity, conductivity, calcium ion, sulfate, nitrate, silicate or chloride. Taxa were distributed at random among the sites – there were no statistically significant pairs of taxa occurring together or avoiding each other. Multivariate analysis of zooplankton associations showed no evidence of distinct associations that could be used to distinguish lakes from wetlands. Zooplankton community structure appears to be a poor tool for distinguishing between lakes and wetlands, especially at the relatively large scale of Wisconsin (dimension of about 500 km). The data suggest that a small body of water in Wisconsin could be classified as a wetland if it persists in the spring and summer for only about 4 months, and if it is inhabited by Lynceus brachyurus, Eubranchipus bundyi, and if Chaoborus americanus and Chydorus brevilabris are absent.     

Role of Rab5 in insulin receptor-mediated endocytosis and signaling

Activated insulin receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between insulin receptor signaling and endocytosis is not well understood. This study utilizes both overexpression and depletion of Rab5 proteins to show that they play a critical role in both insulin-stimulated fluid phase and receptor-mediated endocytosis. Specifically, Rab5:WT and Rab5:Q79L (a GTP-hydrolysis defective mutant) enhance both types of endocytosis in response to insulin, while Rab5:S34N (a GTP-binding defective mutant) has the opposite effect. Morphological analysis indicates that both Rab5 and insulin receptor are found on early endosomes, but not at the plasma membrane. In addition, expression of Rab5:WT and Rab5:Q79L enhance both Erk1/2 and Akt activation without affecting JN- and p38-kinase activities, while the expression of Rab5:S34N blocks both Erk1/2 and Akt activation. Consistent with these observations, DNA synthesis is also altered by the expression of Rab5:S34N. Taken together, these results demonstrate that Rab5 is required for insulin receptor membrane trafficking and signaling.     

Periaqueductal gray/dorsal raphe dopamine neurons contribute to sex differences in pain-related behaviors

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