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β-Arrestins regulate a Ral-GDS–Ral effector pathway that mediates cytoskeletal reorganization

Nature Cell Biology volume 4, pages 547–555 (2002)Cite this article

Abstract

β-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton. We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a β-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs). Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with β-arrestins. In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, β-arrestin–Ral-GDS protein complexes dissociate and Ral-GDS translocates with β-arrestin from the cytosol to the plasma membrane, resulting in the Ras-independent activation of the Ral effector pathway required for cytoskeletal rearrangement. The subsequent re-association of β-arrestin–Ral-GDS complexes is associated with the inactivation of Ral signalling. Thus, β-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.

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Figure 1: Identification of Ral-GDS as a β-arrestin-interacting protein.
Figure 2: Characterization of fMLP-receptor-induced membrane ruffling.
Figure 3: fMLP-receptor-stimulated membrane ruffling in RBL-2H3 cells.
Figure 4: fMLP-receptor-stimulated binding of 35S-GTP-γS to RalA.
Figure 5: fMLP-receptor-stimulated dissociation of β-arrestin–Ral-GDS protein complexes.
Figure 6: fMLP-receptor-stimulated plasma membrane translocation of GFP–Ral-GDS.
Figure 7: Structure–function analysis of Ral-GDS activity.
Figure 8: A model illustrating the regulation of Ral-GDS by β-arrestins.

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Acknowledgements

We thank C. Kubu, E. Li, C. Godin and A. Shin for technical assistance. We thank L. Limbird for helpful discussions. M.B. is the recipient of a Canadian Institutes of Health Research (CIHR) Fellowship, A.V.B. is the recipient of a Canadian Hypertension Society/CIHR Fellowship, J.M.V. is a CIHR Scholar, R.D.F. holds a Heart and Stroke Foundation of Ontario (HSFO) Career Scientist Award and S.S.G.F. is the recipient of Heart and Stroke Foundation of Canada MacDonald Scholarship, Premier's Research Excellence Award and Canada Research Chair in Molecular Neuroscience. This work was supported by HSFO grant T4987 and CIHR grant MA-15506 to S.S.G.F, CIHR MT-10864 to R.J.R. and CIHR grant 43959 to R.D.F.

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Authors and Affiliations

  1. Cell Biology Research Group, The John P. Robarts Research Institute, The University of Western Ontario 100 Perth Drive, London, N6A 5K8, Ontario, Canada

    Moshmi Bhattacharya, Pieter H. Anborgh, Andy V. Babwah, Lianne B. Dale, Tomas Dobransky, Ross D. Feldman, Joseph M. Verdi, R. Jane Rylett & Stephen S. G. Ferguson

  2. Department of Physiology, The University of Western Ontario 100 Perth Drive, London, N6A 5K8, Ontario, Canada

    Ross D. Feldman, R. Jane Rylett & Stephen S. G. Ferguson

  3. Department of Pharmacology and Toxicology, The University of Western Ontario, 100 Perth Drive, London, N6A 5K8, Ontario, Canada

    Ross D. Feldman, R. Jane Rylett & Stephen S. G. Ferguson

  4. Department of Medicine, The University of Western Ontario 100 Perth Drive, London, N6A 5K8, Ontario, Canada

    Ross D. Feldman

  5. Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, 926 BLSB, Philadelphia, 19107, PA, USA

    Jeffery L. Benovic

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Supplementary information

Supplementary movie

Time-lapse recording of membrane ruffling in response to fMLP receptor activation. Shown are two HEK 293 cells, one cell expresses GFP-tagged fMLP receptor (green) alone and the other cell co-expresses Ds-Red1-tagged Ral-GDS clone 284 (red) with GFP-tagged fMLP receptor. The cells correspond to the micrograph presented in Fig. 2b. Membrane ruffling is initiated in the cell lacking Ds-Red1- tagged Ral-GDS clone 284 by the addition of 100 nM fMLP to the dish. The movie is comprised of a time series of 133 confocal images scanned at 15 s intervals. The footage is repeated twice and the presentation speed is accelerated 60 times normal (1s = 1min). (AVI 4483 kb)

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Bhattacharya, M., Anborgh, P., Babwah, A. et al. β-Arrestins regulate a Ral-GDS–Ral effector pathway that mediates cytoskeletal reorganization. Nat Cell Biol 4, 547–555 (2002). https://doi.org/10.1038/ncb821

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