GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production

Abstract

GRB2 is an adaptor protein required for facilitating cytoplasmic signaling complexes from a wide array of binding partners. GRB2 has been reported to exist in either a monomeric or dimeric state in crystal and solution. GRB2 dimers are formed by the exchange of protein segments between domains, otherwise known as “domain-swapping”. Swapping has been described between SH2 and C-terminal SH3 domains in the full-length structure of GRB2 (SH2/C–SH3 domain-swapped dimer), as well as between α-helixes in isolated GRB2 SH2 domains (SH2/SH2 domain-swapped dimer). Interestingly, SH2/SH2 domain-swapping has not been observed within the full-length protein, nor have the functional influences of this novel oligomeric conformation been explored. We herein generated a model of full-length GRB2 dimer with an SH2/SH2 domain-swapped conformation supported by in-line SEC–MALS–SAXS analyses. This conformation is consistent with the previously reported truncated GRB2 SH2/SH2 domain-swapped dimer but different from the previously reported, full-length SH2/C-terminal SH3 (C–SH3) domain-swapped dimer. Our model is also validated by several novel full-length GRB2 mutants that favor either a monomeric or a dimeric state through mutations within the SH2 domain that abrogate or promote SH2/SH2 domain-swapping. GRB2 knockdown and re-expression of selected monomeric and dimeric mutants in a T cell lymphoma cell line led to notable defects in clustering of the adaptor protein LAT and IL-2 release in response to TCR stimulation. These results mirrored similarly-impaired IL-2 release in GRB2-deficient cells. These studies show that a novel dimeric GRB2 conformation with domain-swapping between SH2 domains and monomer/dimer transitions are critical for GRB2 to facilitate early signaling complexes in human T cells.

Grb2 Y160F mutant mimics the wild-type monomeric state dynamics and the monomer-dimer equilibrium

“…Curiously, in contrast to the wild-type, these new conformations reveal that the SH3 domains move apart and do not interact with each other, rotating in opposite directions towards the SH2 domain. This result agrees with the multiple conformations in which both SH3 domains take different positions and orientations in relation to the SH2 domain [14], [17].…”

“…The minor population of Grb2 Y160F exhibited a diameter of 6.3 (± 0.3) nm and an estimated molecular weight of 50 (± 5) kDa, resembling Grb2 WT , which had a diameter of 6.5 (± 0.3) nm and an estimated molecular weight of 53.5 (± 5) kDa. These findings are consistent with the expected values for the dimeric state of Grb2 and suggest the existence of other dimer interfaces, such as the dimerization mediated by SH2 domain-swapping [14]. In addition, the faster decay of the correlation coefficient for Grb2 Y160F (Figure 3B) indicates a predominant monomeric state in solution for the mutant protein despite being able to dimerize.…”

Stimulation of the catalytic activity of the tyrosine kinase Btk by the adaptor protein Grb2

“…In T cells, a protein closely related to Grb2, GADS (Grb2-related adaptor downstream of Shc), works together with Grb2 to crosslink scaffolding proteins and enzymes downstream of the TCR (T-cell receptor) ( Liu et al, 1999 ). Grb2 is also reported to be capable of dimerization, and dimeric Grb2 hinders basal signaling of the fibroblast growth factor receptor 2 (FGFR2), thereby tuning the activity of the receptor ( Lin et al, 2012 ; Sandouk et al, 2023 ).…”