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CRACR2a is a calcium-activated dynein adaptor protein that regulates endocytic traffic.

ABSTRACT: Cytoplasmic dynein is a minus end-directed microtubule motor that transports intracellular cargoes. Transport is initiated by coiled-coil adaptors that (a) join dynein and its cofactor dynactin into a motile complex and (b) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, that have a coiled-coil adaptor domain, a pair of EF-hands, and a Rab GTPase fused into a single polypeptide. CRACR2a-mediated, but not Rab45-mediated, dynein motility is activated by calcium in vitro. In Jurkat T cells, elevation of intracellular calcium activates CRACR2a-mediated dynein transport. We further found that T cell receptor activation induces the formation of CRACR2a puncta at the plasma membrane, which initially associate with the actin cortex and subsequently detach and travel along microtubules, suggestive of an endocytic process. These results provide the first examples of Rab GTPases that directly act as dynein adaptors and implicate CRACR2a-dynein in calcium-regulated endocytic trafficking.

SUBMITTER: Wang Y

PROVIDER: S-EPMC6504896 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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CRACR2a is a calcium-activated dynein adaptor protein that regulates endocytic traffic.

Wang Yuxiao Y Huynh Walter W Skokan Taylor D TD Lu Wen W Weiss Arthur A Vale Ronald D RD

The Journal of cell biology 20190227 5

Cytoplasmic dynein is a minus end-directed microtubule motor that transports intracellular cargoes. Transport is initiated by coiled-coil adaptors that (a) join dynein and its cofactor dynactin into a motile complex and (b) interact with a cargo-bound receptor, which is frequently a Rab GTPase on an organelle. Here, we report two novel dynein adaptors, CRACR2a and Rab45, that have a coiled-coil adaptor domain, a pair of EF-hands, and a Rab GTPase fused into a single polypeptide. CRACR2a-mediated ...[more]

PMID: 30814157

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Project description:Many adaptor proteins involved in endocytic cargo transport exhibit additional functions in other cellular processes which may be either related to or independent from their trafficking roles. The endosomal adaptor protein Tollip is an example of such a multitasking regulator, as it participates in trafficking and endosomal sorting of receptors, but also in interleukin/Toll/NF-κB signaling, bacterial entry, autophagic clearance of protein aggregates and regulation of sumoylation. Here we describe another role of Tollip in intracellular signaling. By performing a targeted RNAi screen of soluble endocytic proteins for their additional functions in canonical Wnt signaling, we identified Tollip as a potential negative regulator of this pathway in human cells. Depletion of Tollip potentiates the activity of β-catenin/TCF-dependent transcriptional reporter, while its overproduction inhibits the reporter activity and expression of Wnt target genes. These effects are independent of dynamin-mediated endocytosis, but require the ubiquitin-binding CUE domain of Tollip. In Wnt-stimulated cells, Tollip counteracts the activation of β-catenin and its nuclear accumulation, without affecting its total levels. Additionally, under conditions of ligand-independent signaling, Tollip inhibits the pathway after the stage of β-catenin stabilization, as observed in human cancer cell lines, characterized by constitutive β-catenin activity. Finally, the regulation of Wnt signaling by Tollip occurs also during early embryonic development of zebrafish. In summary, our data identify a novel function of Tollip in regulating the canonical Wnt pathway which is evolutionarily conserved between fish and humans. Tollip-mediated inhibition of Wnt signaling may contribute not only to embryonic development, but also to carcinogenesis. Mechanistically, Tollip can potentially coordinate multiple cellular pathways of trafficking and signaling, possibly by exploiting its ability to interact with ubiquitin and the sumoylation machinery.

Project description:We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4-64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5'-[?-thio]triphosphate (GTP?S). Block by GTP?S is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTP?S. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief ?-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.

Dataset Information

CRACR2a is a calcium-activated dynein adaptor protein that regulates endocytic traffic.

Publications

CRACR2a is a calcium-activated dynein adaptor protein that regulates endocytic traffic.

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