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Rab GTPases pp 331-354 | Cite as

Quantitative Bead-Based Flow Cytometry for Assaying Rab7 GTPase Interaction with the Rab-Interacting Lysosomal Protein (RILP) Effector Protein

  • Jacob O. Agola
  • Daniel Sivalingam
  • Daniel F. Cimino
  • Peter C. Simons
  • Tione Buranda
  • Larry A. Sklar
  • Angela Wandinger-NessEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1298)

Abstract

Rab7 facilitates vesicular transport and delivery from early endosomes to late endosomes as well as from late endosomes to lysosomes. The role of Rab7 in vesicular transport is dependent on its interactions with effector proteins, among them Rab-interacting lysosomal protein (RILP), which aids in the recruitment of active Rab7 (GTP-bound) onto dynein–dynactin motor complexes to facilitate late endosomal transport on the cytoskeleton. Here we detail a novel bead-based flow cytometry assay to measure Rab7 interaction with the Rab-interacting lysosomal protein (RILP) effector protein and demonstrate its utility for quantitative assessment and studying drug–target interactions. The specific binding of GTP-bound Rab7 to RILP is readily demonstrated and shown to be dose-dependent and saturable enabling K d and B max determinations. Furthermore, binding is nearly instantaneous and temperature-dependent. In a novel application of the assay method, a competitive small molecule inhibitor of Rab7 nucleotide binding (CID 1067700 or ML282) is shown to inhibit the Rab7–RILP interaction. Thus, the assay is able to distinguish that the small molecule, rather than incurring the active conformation, instead ‘locks’ the GTPase in the inactive conformation. Together, this work demonstrates the utility of using a flow cytometry assay to quantitatively characterize protein–protein interactions involving small GTPases and which has been adapted to high-throughput screening. Further, the method provides a platform for testing small molecule effects on protein–protein interactions, which can be relevant to drug discovery and development.

Key words

Ras superfamily Rab Protein–protein interaction Guanine nucleotide binding GTP hydrolysis GTPase effector Quantitative flow cytometry Glutathione-S-transferase (GST) assay Drug discovery HTS—High-throughput screening Structure–activity relationship (SAR), G-Trap assay 

Notes

Acknowledgements

This work was generously supported by National Science Foundation (MCB0956027) and the National Institutes of Health (R21NS7740241) to AWN and (P30CA1181000, U54MH074425, and U54MH084690) to LAS. DS was supported as a visiting MARC scholar (T34 GM008395, PI Zavala, CSUN) and as a summer intern (ASERT IRACDA K12 GM088021, PI Wandinger-Ness). We thank Ms. Janet Kelly for administrative support. We also acknowledge Elsa Romero and Patricia Jim for technical support. Small molecule screening was performed in the NMMLSC/UNMCMD and flow cytometry assays were conducted in the Flow Cytometry Shared Resource Center supported by the University of New Mexico Cancer Center (P30 CA11810).

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Jacob O. Agola
    • 1
    • 2
    • 3
  • Daniel Sivalingam
    • 4
    • 5
  • Daniel F. Cimino
    • 2
    • 6
  • Peter C. Simons
    • 1
    • 2
    • 7
  • Tione Buranda
    • 1
    • 2
  • Larry A. Sklar
    • 1
    • 2
    • 7
  • Angela Wandinger-Ness
    • 1
    • 2
    Email author
  1. 1.Department of PathologyUniversity of New Mexico School of MedicineAlbuquerqueUSA
  2. 2.Cancer CenterUniversity of New Mexico School of MedicineAlbuquerqueUSA
  3. 3.Center for Micro-Engineered Materials, Department of Chemical and Biological Engineering, School of EngineeringUniversity of New MexicoAlbuquerqueUSA
  4. 4.Department of BiologyCalifornia State UniversityNorthridgeUSA
  5. 5.Department of Neurobiology, David Geffen School of MedicineUniversity of CaliforniaLos AngelesUSA
  6. 6.Department of Cell Biology and PhysiologyUniversity of New Mexico School of MedicineAlbuquerqueUSA
  7. 7.Center for Molecular DiscoveryUniversity of New Mexico School of MedicineAlbuquerqueUSA

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