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Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1

Nature Structural & Molecular Biology volume 13, pages 798–805 (2006)Cite this article

Abstract

Receptor oligomerization is vital for activating intracellular signaling, in part by initiating events that recruit effector and adaptor proteins to sites of active signaling. Whether these distal molecules themselves oligomerize is not well appreciated. In this study, we examined the molecular interactions of the adaptor protein GRB2. In T cells, the SH2 domain of GRB2 binds phosphorylated tyrosines on the adaptor protein LAT and the GRB2 SH3 domains associate with the proline-rich regions of SOS1 and CBL. Using biochemical and biophysical techniques in conjunction with confocal microscopy, we observed that the simultaneous association of GRB2, via its SH2 and SH3 domains, with multivalent ligands led to the oligomerization of these ligands, which affected signaling. These data suggest that multipoint binding of distal adaptor proteins mediates the formation of oligomeric signaling clusters vital for intracellular signaling.

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Figure 1: Sedimentation velocity analysis of the interaction of GRB2 and SOS1.
Figure 2: Reverse ITC injections of GRB2 and SOS1.
Figure 3: Relative expression of endogenous GRB2 and SOS1 in Jurkat E6.1 cells.
Figure 4: Sedimentation velocity analysis of the interaction of GRB2, SOS1 and LAT phosphopeptides.
Figure 5: Localization of LAT mutants to signaling clusters.
Figure 6: Effects of expression of SOS1 proline-rich fragments on localization of LAT and PLCG1 to signaling clusters and on TCR-induced Ca2+ influx.
Figure 7: Schematic representation of LAT oligomerization.

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Acknowledgements

We thank D. Piston (Vanderbilt) for providing the mCerulean-C1 vector, W.Y. Langdon (University of Western Australia) for providing the CBL complementary DNA, D. Lowy (National Cancer Institute) for providing the SOS1 cDNA, P.P. Di Fiore (FIRC Institute for Molecular Oncology) for providing the GRB2 cDNA, A. Weiss (University of California at San Francisco) for providing the JCaM 2.5 Jurkat cell line and P. Schwartzberg and J. O'Shea for helpful discussions. This research was supported, in part, by the Intramural Research Program of the US National Institutes of Health, National Cancer Institute, Center for Cancer Research.

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Author notes

  1. Jon C D Houtman

    Present address: Department of Microbiology, University of Iowa, Iowa City, Iowa, 52242, USA

Authors and Affiliations

  1. Laboratory of Cellular and Molecular Biology Division of Bioengineering and Physical Science, ORS, OD, US National Institutes of Health, Bethesda, 20892, Maryland, USA

    Jon C D Houtman, Mira Barda-Saad, Alex Braiman, Brent Bowden & Lawrence E Samelson

  2. Division of Bioengineering and Physical Science, Laboratory of Cell Biology, National Cancer Institute, ORS, OD, US National Institutes of Health, Bethesda, 20892, Maryland, USA

    Hiroshi Yamaguchi & Ettore Appella

  3. Division of Bioengineering and Physical Science, Protein Biophysics Resource, ORS, OD, US National Institutes of Health, Bethesda, 20892, Maryland, USA

    Peter Schuck

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Contributions

J.C.D.H. contributed to ITC analysis, protein purification, immunoblotting, project concept and manuscript preparation; H.Y. contributed to peptide synthesis; M.B.-S. contributed to imaging analysis; A.B. contributed to Ca2+ influx analysis; B.B. contributed to protein purification; E.A. contributed to peptide synthesis and project concept; P.S. contributed to analytical ultracentrifugation and ITC analyses, project concept and manuscript preparation; and L.E.S. contributed to overall project guidance, project concept and manuscript preparation.

Corresponding author

Correspondence to Lawrence E Samelson.

Supplementary information

Supplementary Fig. 1

ITC analysis of the interaction of Grb2 with Sos1 or LAT. (PDF 32 kb)

Supplementary Fig. 2

SV analysis of the interaction of Grb2, Sos1 and LAT. (PDF 65 kb)

Supplementary Fig. 3

Raw SV profiles of the interaction of Grb2, Sos1 and LAT. (PDF 78 kb)

Supplementary Fig. 4

Effects of LAT mutations on the phosphorylation of signaling proteins. (PDF 31 kb)

Supplementary Methods (PDF 347 kb)

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Houtman, J., Yamaguchi, H., Barda-Saad, M. et al. Oligomerization of signaling complexes by the multipoint binding of GRB2 to both LAT and SOS1. Nat Struct Mol Biol 13, 798–805 (2006). https://doi.org/10.1038/nsmb1133

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