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

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.

Regulated vesicle fusion generates signaling nanoterritories that control T cell activation at the immunological synapse

“…Transfection efficiency was evaluated by flow cytometry. 2011) put forward a role for LAT vesicles in TCR signaling, challenging previous studies that demonstrated the requirement of LAT plasma membrane oligomerization and cis phosphorylation (Houtman et al, 2006) for TCR signal transduction. Moreover, Larghi et al (2013) very recently proposed that a VAMP7-mediated LAT vesicle docking to the synapse was responsible for TCR signaling.…”

“…Our data are compatible with a model whereby specialized intracellular calcium microdomains (Wei et al, 2009) and the presence of SNAREs in particular plasma membrane subdomains would locally target LAT vesicle fusion (Video 1). Within those subdomains, delivered LAT would rapidly cluster, as a direct consequence of LAT oligomerization (Houtman et al, 2006). It remains to be addressed whether elongated pLAT clusters originate directly from vesicular LAT, or if they originate because of lateral movement of the plasma membrane after vesicle fusion.…”

A new adaptive grid-size algorithm for the simulation of sedimentation velocity profiles in analytical ultracentrifugation

“…It has significantly improved detection limits and resolution, and our ability to quantitatively describe complex sedimentation processes of macromolecular mixtures. In particular, the convenient use of LE solutions as model function in non-linear regression and/or as kernels in Fredholm integral equations enabled many new techniques, including direct boundary modeling with distributions of LE solutions, and direct boundary modeling with systems of LEs with chemically reacting species, which found wide-spread application in physical biochemistry, for example, in the study of protein-protein interactions [46,49,59,71], the detection of immunogenic oligomeric species of protein pharmaceuticals [10][11][12][13], global multi-signal sedimentation velocity analysis of reversible multi-protein complexes [23,57,[72][73][74], and other applications [52].…”

Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis

“…In fact, LAT oligomerization and SOSmediated Ras activation, which are Grb2 dependent (8,10,28), were described to be important for b-selection at the DN3 stage. This was shown in SOS1 2/2 mice that were reconstituted with an SOS mutant, which are not capable of activating Ras, or an SOS1-SH2 recombinant protein that is unable to cluster phosphorylated LAT.…”

“…In this model, Grb2 binds to phosphorylated tyrosine residues of LAT via its SH2 domain, whereas its SH3 domains bind to SOS1, Cbl, or Gab1. Because LAT, SOS1, Cbl, and Gab1 have more than one Grb2 binding site, a large multiprotein cluster can be formed (8)(9)(10).…”