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Published on: October 30, 2012
Dimerization in the Grb7 protein
Tabitha A Peterson1, Renee L Benallie, Andrew M Bradford
1Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, NM 88003, USA.
Abstract:
In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor-bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7-Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic mutant (Y80E-Grb7-SH2) is largely dimerization deficient and binds a tyrosine-phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild-type SH2 domain. Another dimerization-deficient mutant (F99R-Grb7-SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E-Grb7-SH2 and F99R-Grb7-SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type-Grb7-SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7-SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways.
Insights
Growth factor receptor-bound protein 7 (Grb7) dimerization is crucial for binding receptor tyrosine kinases (RTKs). Phosphorylation of Grb7
Area of Science:
- Molecular Biology
- Cell Signaling
- Protein Structure and Function
Background:
- Previous studies demonstrated that the tyrosine phosphorylation of Grb7 impacts its interactions with FHL2 and human HS-1-associated protein-1.
- Receptor tyrosine kinases (RTKs) dimerization is a known regulatory mechanism in signaling pathways.
Purpose of the Study:
- To investigate the role of dimerization in the Grb7 Src homology 2 (SH2) domain.
- To determine how Grb7 dimerization affects its binding to phosphorylated tyrosine peptide ligands, specifically from the RTK erbB2.
Main Methods:
- Utilized circular dichroism and nuclear magnetic resonance to assess structural integrity and thermal stability of Grb7 SH2 domain mutants.
- Created and analyzed tyrosine phosphorylation-mimic (Y80E) and dimerization-deficient (F99R) Grb7 SH2 domain mutants.
- Measured binding thermodynamics of wild-type and mutant Grb7 SH2 domains to a phosphorylated erbB2 peptide.
Main Results:
- Tyrosine phosphorylation-mimic and dimerization-deficient Grb7 SH2 mutants exhibited impaired dimerization.
- Mutants bound the phosphorylated erbB2 peptide with altered thermodynamic characteristics compared to wild-type.
- While structurally intact, both mutants showed reduced thermal stability.
- Grb7 SH2 domain phosphorylation may regulate Grb7 dimerization.
Conclusions:
- Grb7 dimerization is critical for its structural integrity and ligand binding affinity.
- Phosphorylation of tyrosine residues within the Grb7 SH2 domain likely controls its dimerization.
- Dimerization represents a potential regulatory mechanism for Grb7 binding to RTKs like erbB2, impacting downstream signaling.
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