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Structure of the p115RhoGEF rgRGS domain–Gα13/i1 chimera complex suggests convergent evolution of a GTPase activator

Nature Structural & Molecular Biology volume 12, pages 191–197 (2005)Cite this article

Abstract

p115RhoGEF, a guanine nucleotide exchange factor (GEF) for Rho GTPase, is also a GTPase-activating protein (GAP) for G12 and G13 heterotrimeric Gα subunits. The GAP function of p115RhoGEF resides within the N-terminal region of p115RhoGEF (the rgRGS domain), which includes a module that is structurally similar to RGS (regulators of G-protein signaling) domains. We present here the crystal structure of the rgRGS domain of p115RhoGEF in complex with a chimera of Gα13 and Gαi1. Two distinct surfaces of rgRGS interact with Gα. The N-terminal βN–αN hairpin of rgRGS, rather than its RGS module, forms intimate contacts with the catalytic site of Gα. The interface between the RGS module of rgRGS and Gα is similar to that of a Gα–effector complex, suggesting a role for the rgRGS domain in the stimulation of the GEF activity of p115RhoGEF by Gα13.

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Figure 1: Characterization of Gα13/Gαi1 chimeras.
Figure 2: The structure of rgRGS–Gα13/i-5 complex.
Figure 3: Electrostatic potentials of the rgRGS–Gα13/i-5 complex.
Figure 4: The interface between the N-terminal subdomain of rgRGS and Gα13/i-5.
Figure 5: The interface between the RGS subdomain of rgRGS and Gα13/i-5.

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References

  1. Kozasa, T. et al. p115 RhoGEF, a GTPase-activating protein for Gα12 and Gα13. Science 280, 2109–2111 (1998).

    Article  CAS  Google Scholar 

  2. Hart, M.J. et al. Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Gα13. Science 280, 2112–2114 (1998).

    Article  CAS  Google Scholar 

  3. Sah, V.P., Seasholtz, T.M., Sagi, S.A. & Brown, J.H. The role of Rho in G protein–coupled receptor signal transduction. Annu. Rev. Pharmacol. Toxicol. 40, 459–489 (2000).

    Article  CAS  Google Scholar 

  4. Buhl, A.M., Johnson, N.L., Dhanasekaran, N. & Johnson, G.L. Gα12 and Gα13 stimulate Rho-dependent stress fiber formation and focal adhesion assembly. J. Biol. Chem. 270, 24631–24634 (1995).

    Article  CAS  Google Scholar 

  5. Gohla, A., Harhammer, R. & Schultz, G. The G-protein G13 but not G12 mediates signaling from lysophosphatidic acid receptor via epidermal growth factor receptor to Rho. J. Biol. Chem. 273, 4653–4659 (1998).

    Article  CAS  Google Scholar 

  6. Cerione, R.A. & Zheng, Y. The Dbl family of oncogenes. Curr. Opin. Cell Biol. 8, 216–222 (1996).

    Article  CAS  Google Scholar 

  7. Ross, E.M. & Wilkie, T.M. GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. Annu. Rev. Biochem. 69, 795–827 (2000).

    Article  CAS  Google Scholar 

  8. Zheng, B., De Vries, L. & Gist Farquhar, M. Divergence of RGS proteins: evidence for the existence of six mammalian RGS subfamilies. Trends. Biochem. Sci. 24, 411–414 (1999).

    Article  CAS  Google Scholar 

  9. Tesmer, J.J.G., Berman, D.M., Gilman, A.G. & Sprang, S.R. Structure of RGS4 bound to AIF4−-activated Giα1: stabilization of the transition state for GTP hydrolysis. Cell 89, 251–261 (1997).

    Article  CAS  Google Scholar 

  10. Berman, D.M., Wilkie, T.M. & Gilman, A.G. GAIP and RGS4 are GTPase-activating proteins (GAPs) for the Gi subfamily of G protein α-subunits. Cell 86, 445–452 (1996).

    Article  CAS  Google Scholar 

  11. Druey, K.M. & Kehrl, J.H. Inhibition of regulator of G protein signaling function by two mutant RGS4 proteins. Proc. Natl. Acad. Sci. USA 94, 12851–12856 (1997).

    Article  CAS  Google Scholar 

  12. Srinivasa, S.P., Watson, N., Overton, M.C. & Blumer, K.J. Mechanism of RGS4, a GTPase-activating protein for G protein α-subunits. J. Biol. Chem. 273, 1529–1533 (1998).

    Article  CAS  Google Scholar 

  13. Posner, B.A., Mukhopadhyay, S., Tesmer, J.J., Gilman, A.G. & Ross, E.M. Modulation of the affinity and selectivity of RGS protein interaction with G α-subunits by a conserved asparagine/serine residue. Biochemistry 38, 7773–7779 (1999).

    Article  CAS  Google Scholar 

  14. Slep, K.C. et al. Structural determinants for regulation of phosphodiesterase by a G protein at 2.0 Å. Nature 409, 1071–1077 (2001).

    Article  CAS  Google Scholar 

  15. Fukuhara, S., Chikumi, H. & Gutkind, J.S. Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G (12) family to Rho. FEBS Lett. 485, 183–188 (2000).

    Article  CAS  Google Scholar 

  16. Whitehead, I.P. et al. Expression cloning of lsc, a novel oncogene with structural similarities to the Dbl family of guanine nucleotide exchange factors. J. Biol. Chem. 271, 18643–18650 (1996).

    Article  CAS  Google Scholar 

  17. Fukuhara, S., Murga, C., Zohar, M., Igishi, T. & Gutkind, J.S. A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho. J. Biol. Chem. 274, 5868–5879 (1999).

    Article  CAS  Google Scholar 

  18. Jackson, M. et al. Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins. Nature 410, 89–93 (2001).

    Article  CAS  Google Scholar 

  19. Wells, C.D. et al. Mechanisms for reversible regulation between p115 RhoGEF and GTRAP48 and the G12 class of heterotrimeric G proteins. J. Biol. Chem. 277, 1174–1181 (2002).

    Article  CAS  Google Scholar 

  20. Chen, Z., Singer, W.D., Wells, C.D., Sprang, S.R. & Sternweis, P.C. Mapping the Gα13 binding interface of the rgRGS domain of p115RhoGEF. J. Biol. Chem. 278, 9912–9919 (2003).

    Article  CAS  Google Scholar 

  21. Chen, Z., Wells, C.D., Sternweis, P.C. & Sprang, S.R. Structure of the rgRGS domain of p115RhoGEF. Nat. Struct. Biol. 8, 805–809 (2001).

    Article  CAS  Google Scholar 

  22. Longenecker, K.L., Lewis, M.E., Chikumi, H., Gutkind, J.S. & Derewenda, Z.S. Structure of the RGS-like domain from PDZ-RhoGEF: linking heterotrimeric G protein–coupled signaling to Rho GTPases. Structure 9, 559–569 (2001).

    Article  CAS  Google Scholar 

  23. Singer, W.D., Miller, R.T. & Sternweis, P.C. Purification and characterization of the α-subunit of G13. J. Biol. Chem. 269, 19796–19802 (1994).

    CAS  PubMed  Google Scholar 

  24. Wells, C.D., Gutowski, S., Bollag, G. & Sternweis, P.C. Identification of potential mechanisms for regulation of p115 RhoGEF through analysis of endogenous and mutant forms of the exchange factor. J. Biol. Chem. 276, 28897–28905 (2001).

    Article  CAS  Google Scholar 

  25. Berlot, C.H. & Bourne, H.R. Identification of effector-activating residues of Gs α. Cell 68, 911–922 (1992).

    Article  CAS  Google Scholar 

  26. Skiba, N.P., Bae, H. & Hamm, H.E. Mapping of effector binding sites of transducin α-subunit using Gαt/Gαi1 chimeras. J. Biol. Chem. 271, 413–424 (1996).

    Article  CAS  Google Scholar 

  27. Eisenhaure, T.M., Francis, S.A., Willison, L.D., Coughlin, S.R. & Lerner, D.J. The Rho guanine nucleotide exchange factor Lsc homo-oligomerizes and is negatively regulated through domains in its carboxyl terminus that are absent in novel splenic isoforms. J. Biol. Chem. 278, 30975–30984 (2003).

    Article  CAS  Google Scholar 

  28. Chikumi, H. et al. Homo- and hetero-oligomerization of PDZ-RhoGEF, LARG and p115RhoGEF by their C-terminal region regulates their in vivo Rho GEF activity and transforming potential. Oncogene 23, 233–240 (2004).

    Article  CAS  Google Scholar 

  29. Coleman, D.E. et al. Structures of active conformations of Giα1 and the mechanism of GTP hydrolysis. Science 265, 1405–1412 (1994).

    Article  CAS  Google Scholar 

  30. Sondek, J., Lambright, D.G., Noel, J.P., Hamm, H.E. & Sigler, P.B. GTPase mechanism of G proteins from the 1.7 Å crystal structure of transducin α-GDP-AIF4−. Nature 372, 276–279 (1994).

    Article  CAS  Google Scholar 

  31. Tesmer, J.J., Sunahara, R.K., Gilman, A.G. & Sprang, S.R. Crystal structure of the catalytic domains of adenylyl cyclase in a complex with Gsα.GTPγS. Science 278, 1907–1916 (1997).

    Article  CAS  Google Scholar 

  32. Sterne-Marr, R. et al. G protein–coupled receptor kinase 2/Gαq/11 interaction. A novel surface on a regulator of G protein signaling homology domain for binding Gα subunits. J. Biol. Chem. 278, 6050–6058 (2003).

    Article  CAS  Google Scholar 

  33. Horton, R.M., Hunt, H.D., Ho, S.N., Pullen, J.K. & Pease, L.R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68 (1989).

    Article  CAS  Google Scholar 

  34. Lee, E., Linder, M.E. & Gilman, A.G. Expression of G protein α-subunits in Escherichia coli. Methods Enzymol. 237, 146–164 (1994).

    Article  CAS  Google Scholar 

  35. Wells, C., Jiang, X., Gutowski, S. & Sternweis, P.C. Functional characterization of p115 RhoGEF. Methods Enzymol. 345, 371–382 (2002).

    Article  Google Scholar 

  36. Northup, J.K., Smigel, M.D. & Gilman, A.G. The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding. J. Biol. Chem. 257, 11416–11423 (1982).

    CAS  PubMed  Google Scholar 

  37. Adhikari, A. & Sprang, S.R. Thermodynamic characterization of the binding of activator of G protein signaling 3 (AGS3) and peptides derived from AGS3 with Gαi1. J. Biol. Chem. 278, 51825–51832 (2003).

    Article  CAS  Google Scholar 

  38. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  39. Navaza, J. AMoRe: an automated package for molecular replacement. Acta Crystallogr. A 50, 157–163 (1994).

    Article  Google Scholar 

  40. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

  41. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  CAS  Google Scholar 

  42. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  43. Ramachandran, G.N. & Sassiekharan, V. Conformation of polypeptides and proteins. Adv. Protein Chem. 28, 283–437 (1968).

    Article  Google Scholar 

  44. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  45. Guex, N. & Peitsch, M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723 (1997).

    Article  CAS  Google Scholar 

  46. Read, R.J. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A 42, 140–149 (1986).

    Article  Google Scholar 

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Acknowledgements

We thank W. Chen, S. Gutowski, J. Hadas and A. Badejo for technical assistance, and the staff of Advanced Light Source for assistance with data collection. This work was supported by US National Institutes of Health Grants GM31954 (to P.C.S.), and DK46371 (to S.R.S.), by the Robert A. Welch foundation (to P.C.S. and S.R.S.), the Alfred and Mabel Gilman Chair in Molecular Pharmacology (P.C.S.) and the John W. and Rhonda K. Pate Professorship in Biochemistry (to S.R.S.).

Author information

Authors and Affiliations

  1. Department of Biochemistry, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Zhe Chen & Stephen R Sprang

  2. Department of Pharmacology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    William D Singer & Paul C Sternweis

  3. The Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Stephen R Sprang

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  1. Zhe Chen
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Correspondence to Stephen R Sprang.

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Supplementary information

Supplementary Fig. 1

Structural alignment of helical domains of Gα13 and Gαi1. (PDF 526 kb)

Supplementary Fig. 2

The βN–αN segment of rgRGS fails to stimulate the GTPase activity of Gαi1. (PDF 291 kb)

Supplementary Fig. 3

Changes in α3–β5 region of Gα13/i-5 do not make it a better substrate for rgRGS. (PDF 418 kb)

Supplementary Table 1

Basic residues in the helical domain and switch regions unique to the G12 class Gα subunits. (PDF 17 kb)

Supplementary Table 2

List of protein-protein contacts between the rgRGS domain and Gα13/i-5. (PDF 526 kb)

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Chen, Z., Singer, W., Sternweis, P. et al. Structure of the p115RhoGEF rgRGS domain–Gα13/i1 chimera complex suggests convergent evolution of a GTPase activator. Nat Struct Mol Biol 12, 191–197 (2005). https://doi.org/10.1038/nsmb888

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