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

doi:10.1038/s41598-023-30562-7

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

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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 and Jon C D Houtman

Scientific reports, Vol.13(1), 3505

03/02/2023

DOI: 10.1038/s41598-023-30562-7

PMCID: PMC9981690

PMID: 36864087

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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.

Signal Transduction

Adaptor Proteins, Signal Transducing - genetics

Dimerization

GRB2 Adaptor Protein - genetics

Humans

Interleukin-2

Polymers

Scattering, Small Angle

src Homology Domains

T-Lymphocytes

X-Ray Diffraction

Details

Title: Subtitle: GRB2 dimerization mediated by SH2 domain-swapping is critical for T cell signaling and cytokine production
Creators: Aline Sandouk - University of Iowa
Zhen Xu - University of Iowa
Sankar Baruah - Protein and Crystallography Facility, University of Iowa, Iowa City, IA, 52242, USA
Mikaela Tremblay - University of Iowa
Jesse B Hopkins - Argonne National Laboratory
Srinivas Chakravarthy - Argonne National Laboratory
Lokesh Gakhar - University of Iowa
Nicholas J Schnicker - Protein and Crystallography Facility, University of Iowa, Iowa City, IA, 52242, USA
Jon C D Houtman - Department of Microbiology and Immunology, University of Iowa, Iowa City, IA, 52242, USA. Jon-Houtman@uiowa.edu
Resource Type: Journal article
Publication Details: Scientific reports, Vol.13(1), 3505
DOI: 10.1038/s41598-023-30562-7
PMID: 36864087
PMCID: PMC9981690
NLM abbreviation: Sci Rep
ISSN: 2045-2322
eISSN: 2045-2322
Grant note: R21 AI157121 / NIAID NIH HHS P30 CA086862 / NCI NIH HHS T32 GM139776 / NIGMS NIH HHS
Language: English
Date published: 03/02/2023
Academic Unit: Molecular Physiology and Biophysics; Microbiology and Immunology; Medicine Administration; Internal Medicine
Record Identifier: 9984375453302771

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