14-3-3 protein interacts with and affects the structure of RGS domain of regulator of G protein signaling 3 (RGS3)

 

L. Rezabkova1,2, E. Boura1,2, P. Herman3, J. Vecer3, L. Bourova2, P. Svoboda2, M. Sulc4, T. Obsil1,2

 

1Faculty of Science, Charles University, 12843 Prague, Czech Republic

2Institute of Physiology, Academy of Sciences of the Czech Republic, 14220 Prague 4, Czech Republic

3Faculty of Mathematics and Physics, Institute of Physics, Charles University; 12116 Prague, Czech Republic

4Institute of Microbiology, Academy of Sciences of the Czech Republic; 14220 Prague, Czech Republic

Lenka.Rezabkova@seznam.cz

 

 

Regulator of G protein signaling (RGS) proteins share a highly conserved 125-amino-acid large domain that was first identified by its ability to negatively regulate GPCR signaling [1]. Some RGS proteins consist of little more that the RGS domain while others posses long N-terminal or C-terminal extensions (e.g. RGS3) that usually contain additional protein-protein interaction motifs and domains [2]. RGS proteins function as GTPase-activating proteins (GAP) for the Gα subunit of heterotrimeric G proteins. They bind specifically to the GTP-bound forms of Gα and significantly stimulate GTP hydrolysis by stabilizing the transition state.

The activity of RGS proteins is tightly regulated through various mechanisms including phosphorylation and interactions with other proteins. Several RGS proteins, e.g. RGS3, RGS4 and RGS7, have been found to interact with 14-3-3 proteins. 14-3-3 proteins are a family of acidic regulatory proteins that function as molecular scaffolds by modulating the structure of their binding partners. Results published by several groups suggested that the 14-3-3 protein binding to selected RGS proteins decreases their inhibitory effect on G protein signaling presumably by blocking the interaction between RGS and Gα subunit [3,4].

To elucidate the mechanism of 14-3-3 protein-dependent regulation of RGS function, we performed biophysical characterization of interactions between RGS3 and 14-3-3ζ protein. The main goal of our work was to investigate whether the 14-3-3 protein binding affects the conformation of the RGS domain of RGS3 protein. Site-directed mutagenesis was used to generate two single-tryptophan mutants of human RGS3 with the Trp residue located either at the N-terminus close to the phosphorylation site Ser264 (Trp295) or within the RGS domain at the C-terminus of RGS3 molecule (Trp424). Time-resolved tryptophan fluorescence measurements revealed that phosphorylation of Ser264 itself induces significant structural changes in the region surrounding nearby located Trp295 but not Trp424 located within the remote RGS domain. The interaction between the 14-3-3ζ protein and phosphorylated RGS3 induces significant structural changes in the vicinity of both tested tryptophan residues. Moreover, experiments with isolated RGS domain suggest that this domain can, to some extent, interact with 14-3-3 protein in a phosphorylation-independent manner. We also solved a crystal structure of RGS domain of RGS3 at 2.3Å resolution. This structure suggests that 14-3-3-induced conformational change affects the region within the Gα–interacting portion of the RGS domain. This can explain the inhibitory effect of 14-3-3 on GAP activity of RGS3.  

 

References

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3.     T. Benzing, M.B. Yaffe, T. Arnould, L. Sellin, B. Schermer, B. Schilling, R. Schreiber, K. Kunzelmann, G.G. Leparc, E. Kim, G. Walz, J. Biol. Chem., 275, (2000), 28167.

4.     J. Niu, A. Scheschonka, K.M. Druey, A. Davis, E. Reed, V. Kolenko, R. Bodnar, T. Voyno-Yasenetskaya, X. Du, J. Kehrl, N.O. Dulin, Biochem. J., 365, (2002), 677.

 

Acknowledgements.

This work was funded by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620857 and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.