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

Aline Sandouk, Zhen Xu, Sankar Baruah, Mikaela Tremblay, Jesse B Hopkins, Srinivas Chakravarthy, Lokesh Gakhar, Nicholas J Schnicker, Jon C D Houtman

Scientific reports 2023 Mar 02

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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. © 2023. The Author(s).

Citation

Aline Sandouk, Zhen Xu, Sankar Baruah, Mikaela Tremblay, Jesse B Hopkins, Srinivas Chakravarthy, Lokesh Gakhar, Nicholas J Schnicker, Jon C D Houtman. GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production. Scientific reports. 2023 Mar 02;13(1):3505