Structural basis for inhibition of the insulin receptor by the adaptor protein Grb14

Abstract

Grb14, a member of the Grb7 adaptor protein family, possesses a pleckstrin homology (PH) domain, a C-terminal Src homology-2 (SH2) domain, and an intervening stretch of approximately 45 residues known as the BPS region, which is unique to this adaptor family. Previous studies have demonstrated that Grb14 is a tissue-specific negative regulator of insulin receptor signaling and that inhibition is mediated by the BPS region. We have determined the crystal structure of the Grb14 BPS region in complex with the tyrosine kinase domain of the insulin receptor. The structure reveals that the N-terminal portion of the BPS region binds as a pseudosubstrate inhibitor in the substrate peptide binding groove of the kinase. Together with the crystal structure of the SH2 domain, we present a model for the interaction of Grb14 with the insulin receptor, which indicates how Grb14 functions as a selective protein inhibitor of insulin signaling.

Figure 1. Crystal Structure of the Grb14(BPS)-IRK Complex

(A) Ribbon diagram of the complex between the Grb14 BPS region and phosphorylated IRK. The N-terminal lobe of IRK is colored dark gray, the C-terminal lobe is colored light gray, and Grb14(BPS) is colored purple. The catalytic loop of IRK (residues 1130–1137) is colored orange, and the activation loop (residues 1150–1171) is colored green. Select side chains in IRK are shown in ball-and-stick representation. Carbon atoms are colored yellow (in IRK catalytic loop) or green (in IRK activation loop), oxygen atoms are colored red, nitrogen atoms are colored blue, and phosphorus atoms are colored black. The N- and C termini of IRK and the BPS region are labeled (N and C) as are β1, β2, and α1 of BPS and αC in the N-terminal kinase lobe. (B) View of the pseudosubstrate inhibitory region of Grb14(BPS). Superimposed on the Grb14(BPS)-IRK structure are IRS1-derived substrate peptides from the structures of peptide bound IRK (colored black) (Hubbard, 1997) and peptide bound IGF1 receptor kinase (colored blue) (Favelyukis et al., 2001). In addition to the substrate tyrosine Y(0), the side chains of residues in common with the BPS pseudosubstrate region are shown: Met(+3) of IRS1 ⁷²⁷YMNM bound to IRK (colored gray) and Val(+1) and Phe(+5) of IRS1 ⁸⁹⁵YVNIEF bound to the IGF1 receptor kinase (colored light blue). The dashed black line represents a hydrogen bond between D1132 and the superimposed Y(0) from IRS1 ⁷²⁷YMNM. A semitransparent surface is shown for IRK. The beginning of the BPS α helix is semitransparent to more easily view the pseudo-substrate region beneath. The viewing angle is the same as in (A). (C) Stereo view of the interactions between Grb14(BPS) and IRK. All of the side chains of the BPS region are shown as well as select side chains of IRK. Hydrogen bonds/salt bridges are represented by dashed black lines. The coloring and viewing angle are the same as in (A). (D) Stereo view of a 2F_o − F_c electron density map (3.2 Å, 1.0 σ contour) in the Grb14(BPS)-IRK interface. The viewing angle is 90° from that in (C), on the right. Carbon atoms are colored pink (BPS), green (IRK activation loop), yellow (IRK catalytic loop), or gray (rest of IRK), and phosphorus atoms are colored white. Hydrogen bonds/salt bridges are represented by dashed white lines. Figures 1, 2, and 4 were rendered with PyMOL software (http://pymol.sourceforge.net).

Figure 2. Crystal Structure of the Grb14 SH2 Domain

(A) Ribbon diagram of the Grb14 SH2 domain. The secondary structure elements are labeled. Shown in ball-and-stick representation are phosphotyrosine-interacting residues Arg466, Lys484, and Lys486. (B) Ribbon diagram of the noncrystallographic Grb14 SH2 dimer. The two protomers are colored cyan and orange. The viewing angle is 90° from that in (A), as indicated. The noncrystallographic 2-fold axis is vertical, in the plane of the page. Ile491 in βD, shown with a van der Waals surface, is a key residue in this dimer interface. (C) Ribbon diagram of the crystallographic Grb14 SH2 dimer. The crystallographic 2-fold axis is perpendicular to the page. The key residue in this dimer interface is Phe519 in αB, shown with a van der Waals surface.

Figure 3. Interaction between Grb14 and the Insulin Receptor in Cells

(A) Myc-tagged Grb14 (wt or mutant) was transiently transfected into CHO-IR cells, and coimmunoprecipitations were performed after treatment with 20 nM insulin for 5 min. The lysates were immunoblotted with an antibody to the insulin receptor β subunit to confirm that approximately equal amounts of insulin receptor were present (bottom). Antibodies used for Western blotting are shown on the right side of the blots, and protein identification is supplied on the left side. (B) Same as in (A), except the Grb14 mutations are to the equivalent residues in Grb7. (C) CHO-IR cells were transiently transfected with plasmids coding for Myc-Grb14 (wt or mutant) or with empty vector pRK5, as indicated. Cells were stimulated with 1 nM insulin for 0, 5, or 10 min. Immunoblot analyses on the lysates were performed with the antibodies indicated on the right side of the blots. Blots 1, 3, 5, and 7, done with phospho-specific antibodies, show activation levels, and blots 2, 4, 6, 8, and 9 provide loading controls for the corresponding proteins.

Figure 4. Model for the Interaction of Grb14 with the Insulin Receptor

(A) The interaction between the Grb14 SH2 domain and IRK is based on the APS(SH2)-IRK crystal structure (Hu et al., 2003). Coloring is the same as in Figure 1, with the SH2 domain colored cyan. Semitransparent surfaces are shown for IRK and for the BPS region and SH2 domain of Grb14. The 23 residue BPS-SH2 linker (colored orange) was modeled. The dashed red ovals highlight the interactions between Grb14 residues (labeled) and the activation-loop phosphotyrosines. The N terminus of the BPS region and the C terminus of the SH2 domain are labeled N and C, respectively. (B) Model of the Grb14 dimer interacting with the two kinase domains of the insulin receptor β subunits. The dimeric form of the SH2 domain shown is the same as in Figure 2C. In this 2:2 complex, the N termini of the kinase domains (Tyr984, colored green) are 38 Å apart, with 32 residues of juxtamembrane region (not present in figure) linking each kinase domain to its transmembrane helix. The PH domain is shown schematically as interacting with the membrane. The N-terminal Ras-associating domain of Grb14 (see Figure S1A) is not depicted. The model is 2-fold symmetric about a vertical axis perpendicular to the plane of a modeled membrane bilayer, which is colored yellow (carbon atoms) and red (oxygen atoms) and shown to scale.