Dissertation
SH2/SH2-mediated domain-swapped dimerization of GRB2 and its implications for GRB2 function
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2023
DOI: 10.25820/etd.006997
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, in a phenomenon 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 domainswapping between SH2 domains and monomer/dimer transitions are critical for GRB2 to facilitate early signaling complexes in human T cells. We also investigated a novel role for GRB2 in binding DNA and identified preliminary crystallization conditions for further structural determinations around GRB2 and a potential GRB2-DNA complex.
Details
- Title: Subtitle
- SH2/SH2-mediated domain-swapped dimerization of GRB2 and its implications for GRB2 function
- Creators
- Aline Sandouk
- Contributors
- Jon Houtman (Advisor)Gail Bishop (Committee Member)John Colgan (Committee Member)Miles Pufall (Committee Member)Todd Washington (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Immunology
- Date degree season
- Spring 2023
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.006997
- Number of pages
- viii, 142 pages
- Copyright
- Copyright 2023 Aline Sandouk
- Comment
This thesis has been optimized for improved web viewing. If you require the original version, contact the University Archives at the University of Iowa: https://www.lib.uiowa.edu/sc/contact/.
- Language
- English
- Date submitted
- 05/09/2023
- Date approved
- 05/24/2023
- Description illustrations
- illustrations, tables, graphs
- Description bibliographic
- Includes bibliographical references (pages 110-141).
- Public Abstract (ETD)
- GRB2 is a protein required for cell signaling through its interactions with various binding partners. GRB2 occurs as either a single molecule referred to as a monomer or in pairs referred to as dimers, sometimes through the exchange of protein parts in a phenomenon known as "domain-swapping". Swapping has been described between whole domains in the full-length structure of GRB2, as well as between sub-domain structures known as α-helixes. Interestingly, sub-domain swapping has not been observed in the full-length protein, nor have the functional implications of this phenomenon been explored. We generated a model of a full-length GRB2 dimer with a sub-domain-swapped conformation supported by biophysical and biochemical experiments. We validated our model using several novel GRB2 mutants that promote either the GRB2 monomer or dimer. In cells lacking GRB2, we observed defects in signaling complexes needed for normal T cell function following T cell stimulation. These studies show that a novel GRB2 dimer with domain-swapping between sub-domain structures are important features of GRB2’s role in signaling events in human T cells.
- Academic Unit
- Immunology Graduate Program
- Record Identifier
- 9984428938502771
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