Grb2 adaptor undergoes conformational change upon dimerization Caleb B. McDonald a , Kenneth L. Seldeen a , Brian J. Deegan a , Marc S. Lewis b , Amjad Farooq a, * a Department of Biochemistry and Molecular Biology and the UM/Sylvester Braman Family Breast Cancer Institute, Leonard Miller School of Medicine, University of Miami, Gautier Building, Room 214, 1011 NW 15th Street, Miami, FL 33136, USA b Molecular Interactions Resource, Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA article info Article history: Received 5 March 2008 and in revised form 7 April 2008 Available online 14 April 2008 Keywords: Grb2 dimerization Isothermal titration calorimetry Analytical ultra-centrifugation Size-exclusion chromatography Mass spectrometry abstract Grb2 is an adaptor protein that couples activated receptor tyrosine kinases to downstream effector mol- ecules such as Ras and Akt. Despite being a central player in mitogenic signaling and a target for thera- peutic intervention, the role of Grb2 oligomerization in cellular signaling is not well understood. Here, using the techniques of size-exclusion chromatography, mass spectrometry, analytical ultra-centrifuga- tion and isothermal titration calorimetry, we demonstrate that Grb2 exists in monomer–dimer equilib- rium in solution and that the dissociation of dimer into monomers is entropically-driven without an unfavorable enthalpic change at physiological temperatures. Our data indicate that enthalpy and entropy of dimer dissociation are highly temperature-dependent and largely compensate each other resulting in negligible effect of temperature on the overall free energy. From the plot of enthalpy change versus tem- perature, the magnitude of heat capacity change derived is much smaller than that expected from the rather large molecular surfaces becoming solvent-occluded upon Grb2 dimerization, implying that Grb2 monomers undergo conformational rearrangement upon dimerization. 3D structural models of Grb2 dimer and monomers suggest strongly that such conformational rearrangement upon dimerization may arise from domain swapping. Taken together, our study provides novel insights into the role of Grb2 as an adaptor in cellular signaling circuitry and how Grb2 dimerization may impart high fidelity in signal transduction as well as lead to rapid signal amplification upon receptor stimulation. Ó 2008 Elsevier Inc. All rights reserved. Grb2 1 is a ubiquitous component of cell signaling networks that couples activated receptor tyrosine kinases (RTKs) to down- stream effectors and regulators. The critical role of Grb2 in cellular signaling is exquisitely demonstrated through defects in mice em- bryos upon the disruption of grb2 gene [1]. Grb2 is a modular pro- tein comprised of a central SH2 domain flanked between an N- terminal SH3 (nSH3) domain and a C-terminal SH3 (cSH3) domain, giving it an overall modular architecture of nSH3–SH2–cSH3 [2]. Grb2 recognizes activated RTKs by virtue of its SH2 domain to bind to tyrosine-phopshorylated (pY) sequences in the context of the consensus motif pYXN located within the cytoplasmic tails of a diverse array of receptors, including EGF and PDGF receptors [3,4]. Alternatively, Grb2 can also indirectly dock onto activated RTKs through the binding of its SH2 domain to pY sequences with- in the adaptor protein p52Shc [5,6]. Given a much broader spec- trum of RTKs recognized by p52Shc, Grb2 is more often seen to fulfill this latter role instead of directly binding to RTKs upon receptor stimulation. For its part, p52Shc contains at least two well-conserved YXN motifs at positions Y239 and Y317 that are subject to phosphorylation by a diverse array of protein tyrosine kinases, including the Src kinase family, upon its recruitment to receptor tails in response to extracellular mitogenic stimuli [7– 9]. It has been shown that phosphorylation of both Y239 and Y317 in p52Shc is required for efficient recruitment of Grb2 to the inner membrane surface [10]. Could this necessity be ex- plained by the ability of Grb2 to participate in cellular signaling as a dimer? Upon the interaction of Grb2 to RTKs directly or indirectly, the SH3 domains of Grb2 present a placid opportunity for a wide variety of proteins, containing proline-rich sequences, to be re- cruited to the inner membrane surface and thus engage in down- stream cellular signaling cascades. Some of the best characterized downstream partners of Grb2 SH3 domains are the guanine nucleotide exchange factor Sos1 [11,12], the adaptor protein Gab1 [13,14], the endocytic GTPase dynamin1 [15,16], the ubiqui- 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.04.008 * Corresponding author. Fax: +1 305 243 3955. E-mail address: amjad@farooqlab.net (A. Farooq). 1 Abbreviations used: AUC, analytical ultra-centrifugation; EGF, epidermal growth factor; Grb2, growth factor receptor binder 2; ITC, isothermal titration calorimetry; MALDI-TOF, matrix-assisted LASER desorpton/ionization-time of flight; MAPK, mitogen-activated protein kinase; MMCO, molecular mass cut-off; PDGF, platelet- derived growth factor; RTK, receptor tyrosine kinase; SASA, solvent-accessible surface area; SEC, size-exclusion chromatography; SH2, Src homology 2; SH3, Src homology 3; Shc, Src homology containing. Archives of Biochemistry and Biophysics 475 (2008) 25–35 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi