Article | Published:

Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI


The proper function of Rho GTPases requires precise spatial and temporal regulation of effector interactions. Integrin-mediated cell adhesion modulates the interaction of GTP-Rac with its effectors by controlling GTP-Rac membrane targeting. Here, we show that the translocation of GTP-Rac to membranes is independent of effector interactions, but instead requires the polybasic sequence near the carboxyl terminus. Cdc42 also requires integrin-mediated adhesion for translocation to membranes. A recently developed fluorescence resonance energy transfer (FRET)-based assay yields the surprising result that, despite its uniform distribution, the interaction of activated V12-Rac with a soluble, cytoplasmic effector domain is enhanced at specific regions near cell edges and is induced locally by integrin stimulation. This enhancement requires Rac membrane targeting. We show that Rho-GDI, which associates with cytoplasmic GTP-Rac, blocks effector binding. Release of Rho-GDI after membrane translocation allows Rac to bind to effectors. Thus, Rho-GDI confers spatially restricted regulation of Rac–effector interactions.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1

    de Curtis, I. Cell migration: GAPs between membrane traffic and the cytoskeleton. EMBO Rep. 2, 277–281 (2001).

  2. 2

    Hall, A. Rho GTPases and the actin cytoskeleton. Science 279, 509–514 (1998).

  3. 3

    Lauffenburger, D. A. & Horwitz, A. F. Cell migration: a physically integrated molecular process. Cell 84, 359–369 (1996).

  4. 4

    Bishop, A. L. & Hall, A. Rho GTPases and their effector proteins. Biochem. J. 348 Pt 2, 241–255 (2000).

  5. 5

    Olofsson, B. Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling. Cell Signal. 11, 545–554 (1999).

  6. 6

    Seabra, M. C. Membrane association and targeting of prenylated Ras-like GTPases. Cell Signal. 10, 167–172 (1998).

  7. 7

    Keep, N. H. et al. A modulator of rho family G proteins, Rho-GDI, binds these G proteins via an immunoglobulin-like domain and a flexible N-terminal arm. Structure 5, 623–633 (1997).

  8. 8

    del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D. & Schwartz, M. A. Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19, 2008–2014 (2000).

  9. 9

    Symons, M. Adhesion signalling: PAK meets Rac on solid ground. Curr. Biol. 10, R535–R537 (2000).

  10. 10

    Kraynov, V. S. et al. Localized Rac activation dynamics visualized in living cells. Science 290, 333–337 (2000).

  11. 11

    Dharmawardhane, S., Sanders, L. C., Martin, S. S., Daniels, R. H. & Bokoch, G. M. Localization of p21-Activated Kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in stimulated cells. J. Cell Biol. 138, 1–14 (1997).

  12. 12

    Lamarche, N. et al. Rac and cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade. Cell 87, 519–529 (1996).

  13. 13

    Westwick, J. K. et al. Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways. Mol. Cell. Biol. 17, 1324–1335 (1997).

  14. 14

    Price, L. S., Leng, J., Schwartz, M. A. & Bokoch, G. M. Activation of rac and cdc42 by integrins mediates cell spreading. Mol. Biol. Cell 9, 1863–1871 (1998).

  15. 15

    Hancock, J. F., Paterson, H. & Marshall, C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell 63, 133–139 (1990).

  16. 16

    Knaus, U. G., Wang, Y., Reilly, A. M., Warnock, D. & Jackson, J. H. Structural requirements for PAK activation by Rac GTPases. J. Biol. Chem. 273, 21512–21518 (1998).

  17. 17

    Kozma, R., Ahmed, S., Best, A. & Lim, L. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol. Cell. Biol. 15, 1942–1952 (1995).

  18. 18

    Jordan, J. D., Landau, E. M. & Iyengar, R. Signalling networks: the origins of cellular multitasking. Cell 103, 193–200 (2000).

  19. 19

    Teruel, M. N. & Meyer, T. Translocation and reversible localization of signalling proteins: a dynamic future for signal transduction. Cell 103, 181–184 (2000).

  20. 20

    Kiosses, W. B., Shattil, S. J., Pampori, N. & Schwartz, M. A. Rac recruits high-affinity integrin αvβ3 to lamellipodia in endothelial cell migration. Nature Cell Biol. 3, 316–320 (2001).

  21. 21

    Miyamoto, S. et al. Integrin function: molecular hierarchies of cytoskeletal and signalling proteins. J. Cell Biol. 131, 791–805 (1995).

  22. 22

    Schwartz, M. A., Lechene, C. & Ingber, D. E. Insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integrin α5β1, independent of cell shape. Proc. Natl Acad. Sci. USA 88, 7849–7853 (1991).

  23. 23

    Lewis, J. M. & Schwartz, M. A. Mapping in vivo associations of cytoplasmic proteins with integrin β1 cytoplasmic domain mutants. Mol. Biol. Cell 6, 151–160 (1995).

  24. 24

    Bourguignon, L. Y., Zhu, H., Shao, L. & Chen, Y. W. CD44 interaction with tiam1 promotes Rac1 signalling and hyaluronic acid-mediated breast tumor cell migration. J. Biol. Chem. 275, 1829–1838 (2000).

  25. 25

    Woods, A. & Couchman, J. R. Syndecan 4 heparan sulfate proteoglycan is a selectively enriched and widespread focal adhesion component. Mol. Biol. Cell 5, 183–192 (1994).

  26. 26

    Scheffzek, K., Stephan, I., Jensen, O. N., Illenberger, D. & Gierschik, P. The Rac–Rho-GDI complex and the structural basis for the regulation of Rho proteins by Rho-GDI. Nature Struct. Biol. 7, 122–126 (2000).

  27. 27

    Hoffman, G. R., Nassar, N. & Cerione, R. A. Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator Rho-GDI. Cell 100, 345–356 (2000).

  28. 28

    Lian, L. Y. et al. Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor Rho-GDI-1. Structure Fold Des. 8, 47–55 (2000).

  29. 29

    Longenecker, K. et al. How Rho-GDI binds Rho. Acta Crystallogr. D. Biol. Crystallogr. 55, 1503–1515 (1999).

  30. 30

    Read, P. W. et al. Human RhoA/Rho-GDI complex expressed in yeast: GTP exchange is sufficient for translocation of RhoA to liposomes. Protein Sci. 9, 376–386 (2000).

  31. 31

    Dirac-Svejstrup, A. B., Sumizawa, T. & Pfeffer, S. R. Identification of a GDI displacement factor that releases endosomal Rab GTPases from Rab-GDI. EMBO J. 16, 465–472 (1997).

  32. 32

    Chant, J. Cell polarity in yeast. Annu. Rev. Cell Dev. Biol. 15, 365–391 (1999).

  33. 33

    Johnson, D. I. & Pringle, J. R. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J. Cell Biol. 111, 143–152 (1990).

  34. 34

    Leng, J., Klemke, R. L., Reddy, A. C. & Cheresh, D. A. Potentiation of cell migration by adhesion-dependent cooperative signals from the GTPase Rac and Raf kinase. J. Biol. Chem. 274, 37855–37861 (1999).

  35. 35

    Habets, G. G. et al. Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins. Cell 77, 537–549 (1994).

  36. 36

    Michiels, F., Habets, G. G. M., Stam, J. C., vanderKammen, R. A. & Collard, J. G. A function for rac in Tiam1-induced membrane ruffling and invasion. Nature 375, 338–340 (1995).

  37. 37

    Fukumoto, Y. et al. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins. Oncogene 5, 1321–1328 (1990).

  38. 38

    Moriyoshi, K., Richards, L. J., Akazawa, C., O'Leary, D. D. & Nakanishi, S. Labeling neural cells using adenoviral gene transfer of membrane- targeted GFP. Neuron 16, 255–260 (1996).

  39. 39

    del Pozo, M. A., Vicente-Manzanares, M., Tejedor, R., Serrador, J. M. & Sanchez-Madrid, F. Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes. Eur. J. Immunol. 29, 3609–3620 (1999).

  40. 40

    Chamberlain, C. E., Kraynov, V. S. & Hahn, K. M. Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR. Methods Enzymol. 325, 389–400 (2000).

  41. 41

    Glaven, J. A., Whitehead, I., Bagrodia, S., Kay, R. & Cerione, R. A. The Dbl-related protein, Lfc, localizes to microtubules and mediates the activation of Rac signalling pathways in cells. J. Biol. Chem. 274, 2279–2285 (1999).

  42. 42

    Gong, M. C. et al. Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II. Am. J. Physiol. Cell Physiol. 281, C257–C269 (2001).

  43. 43

    Read, P. W. & Nakamoto, R. K. Expression and purification of Rho/Rho-GDI complexes. Methods Enzymol. 325, 15–25 (2000).

  44. 44

    Renshaw, M. W., Ren, X.-D. & Schwartz, M. A. Growth factor activation of MAP kinase requires cell adhesion. EMBO J. 16, 5592–5599 (1997).

  45. 45

    Kiosses, W. B., Daniels, R. H., Otey, C., Bokoch, G. M. & Schwartz, M. A. A function for p21-activated kinase in endothelial cell migration. J. Cell. Biol. 147, 831–844 (1999).

  46. 46

    Chamberlain, C. & Hahn, K. M. Watching proteins in the wild: fluorescence methods to study protein dynamics in living cells. Traffic 1, 755–762 (2000).

  47. 47

    Chong, L. D., Traynor-Kaplan, A., Bokoch, G. M. & Schwartz, M. A. The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells. Cell 79, 507–513 (1994).

Download references


We thank P. Read for providing plasmids for purification of Rho-GDI and prenylated Rac, A. Hall for Rac effector mutants, Y. Takai for pEFBOS-myc–Rho-GDI, J.H. Jackson for pRK5-myc–V12-Rac-6Q, K. Moriyoshi for the GAP-43–GFP plasmid and A. Woods for the 150.9 monoclonal anti-syndecan-4. We also thank C. Chamberlain for technical advice establishing the FRET assays, and S. Shattil and E. Tzima for their critical comments. This work was supported by United States Public Health Service grant RO1 GM47214 (to M.A.S) and GM39434 (to K.M.H). M.A.d.P. was supported by the Human Frontier Science Program (LT0019/1998-M) and then by a Lady Tata Memorial Trust International Award for Research in Leukemia.

Author information

Competing interests

The authors declare no competing financial interests.

Correspondence to Miguel Angel Del Pozo or Martin Alexander Schwartz.

Supplementary information

Supplementary figures

Figure S1 Activated GTPases localize at the basal level of the plasma membrane. Confocal z-section series of cells expressing various GFP fusions from Fig. 2 A and C. (PDF 1139 kb)

Figure S2 Time course of integrin-induced FRET in GFP-V12 Rac cells.

Rights and permissions

Reprints and Permissions

About this article

  • Received

  • Revised

  • Accepted

  • Published

  • Issue Date

  • DOI

Further reading

Figure 1: Rac membrane targeting is independent of effector interactions but requires the C-terminal polybasic sequence.
Figure 2: Integrins regulate Cdc42 membrane targeting similarly to Rac.
Figure 3: FRET analysis demonstrates that GTP-Rac coupling to effectors is locally enhanced in lamellipodia.
Figure 4: GTP-Rac coupling to effectors is locally induced by integrin stimulation and requires Rac membrane targeting.
Figure 5: Rho-GDI blocks GTP-Rac effector coupling.
Figure 6: Model for integrin-regulated localized enhancement of Rac membrane targeting and effector coupling.