Structural Basis for Dimerization of the Grb10 Src Homology 2 Domain

Abstract

Grb7, Grb10, and Grb14 are members of a distinct family of adapter proteins that interact with various receptor tyrosine kinases upon receptor activation. Proteins in this family contain several modular signaling domains including a pleckstrin homology (PH) domain, a BPS (between PH and SH2) domain, and a C-terminal Src homology 2 (SH2) domain. Although SH2 domains are typically monomeric, we show that the Grb10 SH2 domain and also full-length Grb10␥ are dimeric in solution under physiologic conditions. The crystal structure of the Grb10 SH2 domain at 1.65-Å resolution reveals a non-covalent dimer whose interface comprises residues within and flanking the C-terminal ␣ helix, which are conserved in the Grb7/ Grb10/Grb14 family but not in other SH2 domains. Val-522 in the BG loop (BG3) and Asp-500 in the EF loop (EF1) are positioned to interfere with the binding of the P؉3 residue of a phosphopeptide ligand. These structural features of the Grb10 SH2 domain will favor binding of dimeric, turn-containing phosphotyrosine sequences, such as the phosphorylated activation loops in the two ␤ subunits of the insulin and insulin-like growth factor-1 receptors. Moreover, the structure suggests the mechanism by which the Grb7 SH2 domain binds selectively to pTyr-1139 (pYVNQ) in Her2, which along with Grb7 is co-amplified in human breast cancers.Grb10 is a member of a family of adapter proteins including Grb7 and Grb14, which have been identified as putative downstream effectors of receptor tyrosine kinases (1). Proteins in this family contain several modular domains including an Nterminal proline-rich region, a Ras-associated-like domain, a pleckstrin homology (PH) 1 domain, a short region known as the BPS (between PH and SH2) or PIR (phosphorylated insulin receptor-interacting region) domain, and a C-terminal Src homology 2 (SH2) domain. Grb10 has multiple alternatively spliced forms (␣-), which differ in their N-terminal region including the PH domain.The BPS/PIR and SH2 domains of Grb7/Grb10/Grb14 have been implicated in the interaction with activated receptor tyrosine kinases. The BPS domain is a structurally uncharacterized region of ϳ50 residues that is unique to this family of adapter proteins. The SH2 domain, found in many signaling proteins, is a well characterized protein module of ϳ100 residues that binds phosphotyrosine-containing sequences (2, 3). The BPS domains of Grb7/Grb10/Grb14 bind to the phosphorylated insulin and insulin-like growth factor-1 (IGF1) receptors (4 -6). The SH2 domains of Grb7/Grb10/Grb14 also interact with the insulin and IGF1 receptors (4, 5, 7-11) and, in addition, have been shown to mediate the interaction with the epidermal growth factor receptor (4, 12), Her2 (epidermal growth factor receptor family) (13, 14), platelet-derived growth factor receptor-␤ (10, 15, 16), Ret (17), EphB1 (18), Kit (19), Tie2 (20), and fibroblast growth factor receptor-1 (21).The roles of Grb10 family members in growth factor-mediated signaling have not been clearly established. Most studies have investigated...

The PIR domain of Grb14 is an intrinsically unstructured protein: implication in insulin signaling

“…For the two other Grbs, the SH2 interaction can su¡er a mismatch minimizing their action as in the Src structure [43], or they can bind preferentially to pY1158 instead of pY1162pY1163. This model is supported by the recent study of the crystal structure of Grb10Q [44] SH2 domain that appears to be a dimer. It has been proposed that the SH2 dimer binds to the activation loops of the two tyrosine kinase domains of the IR L subunits with pY1158 in the canonical phosphotyrosine binding pocket of each SH2 domain.…”

Tissue-specific regulation and function of Grb10 during growth and neuronal commitment

“…The SH2 domain also interacts with many receptor proteins implicated in cell proliferation, differentiation, and control of the cell cycle, including RET, KIT, MET, PDGFR, EGFR, FLT3, and VEGFR-2 (9,(12)(13)(14)(15)(16). Additionally, the SH2 domain has been implicated in Grb10 homodimerization, which is hypothesized to enable Grb10 to integrate signals from multiple pathways through the formation of larger protein complexes (17).…”

The Insulin Receptor: Both a Prototypical and Atypical Receptor Tyrosine Kinase

“…The phosphorylated activation loop represents an atypical SH2-domain binding site, in that the loop is multiply phosphorylated and is stabilized in a turn-containing (rather than an extended) conformation. Indeed, these SH2 domains possess unique features that allow them to bind efficiently to the phosphorylated activation loop of InsR (Hu et al 2003;Stein et al 2003;Depetris et al 2005). First, they are dimeric.…”