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Structure and Mechanism of the Insulin Receptor Tyrosine Kinase
Chapter 45
Structure and Mechanism of the Insulin Receptor Tyrosine Kinase Stevan R. Hubbard Skirball Institute of Biomolecular Medicine and Department of Pharmacology, New York University School of Medicine, New York, New York
Introduction Insulin activates signaling pathways that control cellular metabolism and growth [1]. The actions of this essential hormone are mediated by the insulin receptor [2, 3], a member of the receptor tyrosine kinase (RTK) family of cell surface receptors. The RTK family also includes, among others, the receptors for insulin-like growth factor-1 (IGF1), epidermal growth factor (EGF), fibroblast growth factors (FGFs), platelet-derived growth factors (PDGFs), nerve growth factor (NGF), and vascular endothelial growth factors (VEGFs). A large family of non-receptor tyrosine kinases also exists, which includes Src-family kinases, Abl kinases, Janus kinases (Jaks), Syk and Zap70, and Fak. Receptor and non-receptor tyrosine kinases are critical components of signaling pathways in metazoans, controlling cellular proliferation, differentiation, migration, and metabolism [4]. In contrast to most RTKs, which are single-chain receptors that are activated by ligand-induced oligomerization, the insulin receptor is an 22 heterotetramer (Figure 45.1), with disulfide linkages between the two -chains and between the - and -chains. Insulin binding to the chains induces a structural rearrangement within the receptor, resulting in autophosphorylation of specific tyrosines in the -chain: Tyr972 in the juxtamembrane region, Tyr1158, Tyr1162, and Tyr1163 in the activation loop in the kinase domain, and Tyr1328 and Tyr1334 in the C-terminal region (Figure 45.1). Phosphorylation of Tyr1162 and Tyr1163 (activation loop) stimulates catalytic activity, and phosphorylated Tyr972 (pTyr972; juxtamembrane region), pTyr1158, and pTyr1162 (activation loop) serve as docking sites for downstream signaling proteins. The functional role of tyrosine phosphorylation in the C-terminal region has not been fully established. Handbook of Cell Signaling, Three-Volume Set 2 ed. Copyright © 2010 Elsevier Inc. All rights reserved.
Because the insulin receptor is a transmembrane protein, structural studies to understand signaling mechanisms have largely been confined to either the extracellular region or the cytoplasmic region. Recently, the crystal structure of the disulfide-linked dimeric ectodomain of the insulin receptor was determined [5], which reveals an antiparallel “inverted V” arrangement, and suggests how insulin might cross link the ectodomain by binding to the first L domain of one chain and to the first fibronectin type III domain on the other chain. Crystal structures of the insulin-receptor kinase domain (IRK) in the cytoplasmic portion of the chain have been determined in different states of phosphorylation [6, 7] and for several mutants [8, 9]. IRK shares a similar overall architecture with protein serine/threonine kinases, with an N-terminal lobe (N lobe) comprising a five-stranded anti-parallel -sheet and one -helix, and a larger C lobe which is mainly -helical (Figure 45.1) [10]. ATP binds in the cleft between the two lobes, and the tyrosine-containing segment of a protein substrate binds in the substrate binding groove in the C lobe. A key mechanism by which the insulin receptor and other tyrosine and serine/threonine kinases regulate catalytic activity is through reversible positioning of their activation loops [11]. The activation loop of the insulin receptor, with three autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163), begins with the protein kinase-conserved 1150DFG motif and ends with tyrosine kinase-conserved Pro1172. In the crystal structure of unphosphorylated (basal) IRK, the activation loop traverses the cleft between the two kinase lobes, with Tyr1162 bound in the active site [6]. In addition to obstructing the substrate binding site, this conformation of the activation loop also obstructs the nucleotide binding site, ensuring that Tyr1162 cannot be phosphorylated in cis. The activation loop is dynamic, however, and upon juxtaposition of the two kinase domains through insulin binding to the 307
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PART | I Initiation: Extracellular and Membrane Events
α α L domain cys-rich domain
β
L domain β fibronectin type III domains
Extracellular 972 Cytoplasmic 1158 1162 1163 1328 1334
juxtamembrane region tyrosine kinase domain
C-terminal tail
Figure 45.1 Overall architecture of the insulin receptor and structure of the tyrosine kinase domain. The -chains are extracellular and the -chains pass through the plasma membrane. The major sites of tyrosine autophosphorylation in the cytoplasmic domain are numbered according to Ebina et al. [2]. There are three fibronectin type III (FnIII) domains in the extracellular portion of the receptor. FnIII-1 is contained in the -chain, as is the N-terminal portion of FnIII-2. The C-terminal portion of FnIII-2 is contained in the -chain, followed by FnIII-3. Solid lines between the -chains and between the - and -chains represent disulfide linkages. On the right, a ribbon diagram of the structure of tris-phosphorylated IRK is shown [7], with the N lobe colored dark gray and the C lobe colored light gray. AMP-PNP is shown in stick representation, as are the three activation-loop phosphotyrosines and the substrate tyrosine.
ectodomain, phosphorylation of the activation loops ensues in trans, resulting in phosphotyrosine-mediated stabilization of a loop configuration that is optimal for catalysis [7].
Protein recruitment to the activated insulin receptor Overview Upon insulin-stimulated activation of its receptor, several signaling proteins are recruited to the receptor, either for signal propagation or for attenuation. Among the proteins recruited for activation of downstream signaling pathways are several adaptor (non-enzymatic) proteins, including the insulin receptor substrate (IRS) proteins, Shc, and SH2B2/ APS. Negative regulators recruited to the receptor include the adaptor protein Grb14 and the protein tyrosine phosphatase PTP1B. Recent structural studies have elucidated the modes of binding between these proteins and the cytoplasmic domain of the insulin receptor. Phosphotyrosine recruitment sites in RTKs usually reside in polypeptide segments that are N-terminal to the kinase domain (juxtamembrane region), or C-terminal to the kinase domain, or in the kinase insert region (between helices D and E). These polypeptide segments are typically
unstructured (non--helix, non--strand) and can easily adopt an extended conformation, which is required for phosphorylation, and, upon phosphorylation, for binding to Src homology-2 (SH2) domains and phosphotyrosinebinding (PTB) domains [12]. A subset of SH2 domains, those contained in SH2B2 and Grb14, specifically binds to the phosphorylated activation loop of the insulin receptor, which represents an unusual SH2-domain binding target because it is multiply phosphorylated and held in a relatively stable, turn-containing (non-extended) conformation. As detailed below, these SH2 domains possess three important properties: (1) they are dimeric, (2) they recognize two phosphotyrosines in the IRK activation loop, and (3) they are inhibited from binding canonical phosphotyrosine sequences.
Recruitment of SH2B2 to the Insulin Receptor SH2B2 is a member of a family of three adaptor proteins, SH2B1–3, previously referred to as SH2B, APS, and Lnk, respectively [13]. These proteins consist of an N-terminal dimerization motif, a pleckstrin homology (PH) domain, an SH2 domain, and polyproline stretches in the N- and C-terminal regions. Recruitment of SH2B2 to the insulin
Chapter | 45 Structure and Mechanism of the Insulin Receptor Tyrosine Kinase
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Figure 45.2 Structure of the SH2B2 SH2 domain bound to IRK [15]. In this 2 : 2 complex, IRK is shown with a semi-transparent molecular surface. The phosphotyrosines in the activation loop (pTyr1158/1162/1163) are labeled. The two protomers of the SH2B2 SH2-domain dimer (in the middle of the figure) are shown in ribbon representation.
receptor results in phosphorylation of a C-terminal tyrosine residue in SH2B2, Tyr618, which then serves as a binding site for the tyrosine-kinase binding (TKB) domain of Cbl [14, 15]. This signaling pathway culminates in activation of TC10, a Rho-family GTPase, which is thought to facilitate glucose uptake in adipocytes [1, 16]. The interaction between SH2B2 and the insulin receptor is mediated by the SH2 domain of SH2B2, which, as first shown by yeast two-hybrid studies, interacts with the phosphorylated activation loop of IRK [17, 18]. A crystal structure of the SH2B2 SH2 domain [15] revealed that it possesses a non-canonical architecture, in which the C-terminal half of the domain forms a long -helix rather than two strands (-strands E and F) and a shorter -helix (-helix B) as found in a typical SH2 domain. This structural rearrangement facilitates formation of a novel SH2-domain dimer, which interacts with the activation loops in the two kinase domains of the insulin receptor (Figure 45.2) [15]. Formation of the dimer thwarts binding to canonical phosphotyrosine sequences containing a hydrophobic residue at the 3 position (relative to the phosphotyrosine), and confers specificity for the (turn-containing) phosphorylated IRK activation loop. Rather than coordinating a single phosphotyrosine, each protomer of the SH2B2 SH2 dimer coordinates two phosphotyrosines, the first (pTyr1158) in the canonical (arginine-containing) phosphate binding pocket, and the second (pTyr1162) by two lysines in -strand D (Figure 45.2). Thus, the SH2B2 SH2 dimer is a quad phosphotyrosine-recognition module. Of note, despite a sequence identity of 79 percent between the SH2 domains of SH2B1 and SH2B2, the SH2B1 SH2 domain is monomeric, which switches its binding preference from the IRK activation loop to pTyr813 of Jak2, a conventional SH2-domain ligand with a 3 hydrophobic residue [19].
Recruitment of SH2B2 to the phosphorylated activation loop of the insulin receptor prolongs receptor activation [20], presumably through protection from dephosphorylation by protein tyrosine phosphatases such as PTP1B, and selects for phosphorylation of Tyr618 near the C-terminus of SH2B2, 130 residues C-terminal to the SH2 domain. Although there are numerous tyrosine residues in SH2B2 (68-kDa protein), Tyr618 is the predominant site of phosphorylation by the insulin receptor, despite its non-optimal substrate sequence (non-YXM, where is hydrophobic and X is any residue). Selectivity for Tyr618 is evidently due to the binding of the SH2B2 SH2 dimer to the kinase activation loops, which facilitates entry of Tyr618, near the C-terminus of SH2B2, into the IRK active site.
Recruitment of IRS1 and IRS2 to the Insulin Receptor The IRS proteins are a family of four to six adaptor molecules that possess N-terminal PH and PTB domains, followed by more than 900 residues containing multiple sites of tyrosine and serine/threonine phosphorylation [21]. Knockout studies in mice have shown that IRS1 and IRS2 are essential for organismal development and glucose disposal [21]. Despite a conserved domain architecture, common sites of phosphorylation, and overlapping tissue expression, the phenotypes for the IRS1 and IRS2 knockout mice are distinct. IRS1/ mice are 40 percent smaller than wild-type littermates, and exhibit insulin resistance in peripheral tissues [22, 23]. IRS2/ mice are only slightly smaller (10 percent) than wild-type, but are insulin resistant and develop type 2 diabetes due to insufficient pancreatic -cell function [24].
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Many of the tyrosine phosphorylation sites in the C-terminal region of IRS1/2 reside in a YXM motif, which, when phosphorylated by the insulin receptor (or IGF1 receptor), serves as a recruitment site for the SH2 domain(s) of phosphatidylinositol 3-kinase (PI-3K) [25]. Activation of PI-3K through engagement of tyrosinephosphorylated IRS1/2 is required for glucose uptake into insulin-responsive cells [26]. Other tyrosine phosphorylation sites in IRS1/2 recruit the adaptor protein Grb2 and the protein tyrosine phosphatase SHP2 [21]. IRS1/2 also possess numerous sites of serine/threonine phosphorylation that, in general, negatively regulate tyrosine phosphorylation, either in the course of normal negative feedback or in pathologic insulin resistance [27]. The tandem PH–PTB domains in the N-terminal regions of these adaptor proteins function to recruit IRS1/2 to the insulin receptor for phosphorylation. The PTB domains bind to pTyr972 and adjacent residues (NPXY motif) in the juxtamembrane region of the insulin (and IGF1) receptor [28, 29]. The PH domains are also important for recruitment of IRS1/2 to the receptor [30,31]. Previous yeast twohybrid studies provided evidence for a second (in addition to the PTB domain) insulin receptor-interacting region in IRS2, which was named the kinase regulatory-loop binding (KRLB) region [32, 33] or receptor binding domain-2 (RBD2) [34]. This region of IRS2 interacts with the kinase domain of the insulin receptor (IRK) in a phosphodependent manner [32, 34]. The KRLB region was roughly mapped to residues 591–733 [32, 34], which starts approximately 300 residues C-terminal to the PTB domain. This region has a high proportion of glycine, serine, and proline residues, i.e., it is likely unstructured, and contains three tyrosine residues in a YXM motif. Although the corresponding region in IRS1 contains considerable sequence similarity, including the three YXM sites, it does not stably interact with IRK [33, 34]. Mutagenesis studies in the KRLB region identified two non-YXM tyrosine residues, Tyr624 and Tyr628 (mouse numbering), which are critical for this region of IRS2 to bind to IRK [33]. The molecular basis for the interaction of the IRS2 KRLB region with the insulin receptor was elucidated through co-crystallization of a 15-residue peptide from the KRLB region (containing Tyr624 and Tyr628) with phosphorylated IRK [35]. The structure revealed that this region of IRS2 binds in the active site of IRK with Tyr628 poised for phosphorylation (Figure 45.3). Biochemical experiments demonstrated that Tyr628 is phosphorylated by IRK, but with a Km(ATP) that is very high compared to that of a YXM substrate (1.6 mM vs 40 M, respectively). In addition, the phosphorylated Tyr628 peptide retains significant binding affinity, which translates into poor substrate turn over. As a consequence, this segment of IRS2 inhibits phosphorylation by the insulin receptor of other tyrosine sites in IRS2. A possible functional role of the KRLB region is to suppress tyrosine phosphorylation of IRS2, such that only
PART | I Initiation: Extracellular and Membrane Events
Figure 45.3 Structure of the KRLB region of IRS2 bound to IRK [35]. IRK is shown with a semi-transparent molecular surface. The side chains of the KRLB peptide (N- and C-termini are labeled) are shown in stick representation. Tyr624 and Tyr628 of the KRLB region are labeled, the latter of which is bound in the IRK active site.
metabolic pathways via PI-3 K are stimulated by insulin and not mitogenic pathways via Grb2/Ras; there are 10 potential PI-3 K recruitment sites in IRS2 versus a single Grb2 site.
Recruitment of Grb14 to the Insulin Receptor Among the negative regulators of insulin signaling, Grb14 is a member of the Grb7/10/14 family of adaptor proteins [36]. Grb14/ mice show tissue-specific (muscle and liver) increases in glucose disposal, and patients with type 2 diabetes have been shown to have increased Grb14 mRNA levels in adipose tissue [37]. Grb7/10/14 proteins possess several signaling modules, including a Ras-associating (RA) domain, a PH domain, and a C-terminal SH2 domain, as well as polyproline sequences. In addition, these proteins contain a 45-residue region known as BPS (between PH and SH2) [38] or PIR (phosphorylated insulin receptorinteracting region) [39], which is unique to this adaptor family. Biochemical studies have demonstrated that the BPS region of Grb7/10/14 is capable of directly inhibiting the catalytic activities of the insulin and IGF1 receptors [40, 41], with a potency rank of Grb14Grb10Grb7 [41]. A crystal structure of the Grb14 BPS region in complex with phosphorylated IRK revealed at least one mechanism by which Grb14 inhibits signaling by the insulin receptor [42]. In the structure (Figure 45.4), the N-terminal portion of the BPS region binds in the substrate binding groove in the C lobe of the kinase domain. A leucine (Leu376)
Chapter | 45 Structure and Mechanism of the Insulin Receptor Tyrosine Kinase
is inserted in the active site rather than a tyrosine, and the hydrophobic residues at the 1, 3, and 5 positions relative to Leu376 mimic peptide–substrate binding. Thus, the Grb14 BPS region acts as a pseudosubstrate inhibitor to suppress insulin-receptor signaling. After the pseudosubstrate segment, the Grb14 BPS region adopts a 16-residue -helix whose residues make interactions with the phosphorylated activation loop. These interactions fortify the BPS–IRK interaction and also provide specificity; the BPS region of Grb14/10 only inhibits the insulin and IGF1 receptors. The BPS -helix ends near the kinase N lobe, which positions the C-terminal SH2 domain (25-residue BPS–SH2 linker) for engagement of pTyr1158 and pTyr1162 in the activation loop [42]. The SH2 domain of Grb14/10 is, like the SH2B2 SH2 domain, dimeric, but in this case the individual protomers possess a canonical architecture. Binding of canonical phosphotyrosine sequences (with 3 hydrophobic residues) is deterred in the Grb14/10 SH2 domain, not by dimer formation (as for SH2B2), but by a valine in the BG loop (BG3) rather than a glycine (as in Src and Abl) [43].
Recruitment of PTP1B to the Insulin Receptor PTP1B is a key negative regulator of insulin signaling in vivo, as shown by gene-deletion studies in mice. Despite the ability of PTP1B to dephosphorylate numerous RTKs in vitro and in cells, the PTP1B/ mice are of normal size (i.e., no overgrowth phenotype) and exhibit two apparent phenotypes: a hypersensitivity to insulin, due to prolonged insulin-receptor phosphorylation, and resistance to weight gain on a high-fat diet [44, 45]. The molecular basis for PTP1B’s specificity towards the insulin receptor has not been fully elucidated. A crystal structure of PTP1B with a phosphorylated peptide representing the IRK activation loop revealed that a phosphotyrosine (pTyr1163) following the substrate phosphotyrosine (pTyr1162) is a specificity determinant [46]. Because several other RTKs (e.g., IGF1 receptor, TrkA-C, FGF receptors), which do not appear to be targets of PTP1B in vivo, contain tandem phosphotyrosines in their activation loops, other specificity determinants must exist. A crystal structure of a PTP1B–IRK complex was determined that revealed a novel, non-catalytic mode of interaction between the two proteins, in which PTP1B binds not to a phosphotyrosine in the IRK activation loop, but rather to the opposite side of the kinase domain (Figure 45.5). This interaction was evidently facilitated by crystallization in ammonium sulfate, which effectively competes with the activation-loop phosphotyrosines for binding in the PTP1B active site. Although this interaction might be dismissed as a crystallization artifact, the PTP1B– IRK interface comprises residues that are highly characteristic for these two proteins, including Tyr152 and Tyr153 of
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Figure 45.4 Structure of the BPS region of Grb14 bound to IRK [35]. IRK is shown with a semi-transparent molecular surface. The BPS region (N- and C-termini are labeled) is shown in ribbon representation. Leu376 (labeled), the pseudosubstrate bound in the IRK active site, is shown in stick representation.
Figure 45.5 Structure of a PTP1B–IRK complex. In this 2 : 2 complex, the IRK dimer is in the center of the figure, and the two PTP1B molecules are on the outside, shown in ribbon representation. The molecular (non-crystallographic) two-fold axis is perpendicular to the page.
PTP1B, which had been previously implicated in the interaction between PTB1B and the insulin receptor [47–49]. Moreover, the highly related IGF1 receptor would be incapable of engaging PTP1B in this manner. Thus, it remains an attractive hypothesis that the interaction observed in the crystal structure represents a recruitment mode of binding to localize PTP1B to the insulin receptor, although biochemical evidence for this hypothesis is still outstanding.
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Prospects An understanding of the signaling mechanisms intrinsic to the insulin receptor is not only of fundamental biochemical interest. The prevalence of non-insulin-dependent diabetes mellitus (NIDDM) in developed countries is increasing at an alarming rate. One therapeutic strategy for NIDDM is to activate or potentiate signaling at the level of the insulin receptor. Indeed, small-molecule activators/potentiators of the insulin receptor that act cytoplasmically have been reported [50, 51]. Knowledge of the structural transitions that underlie insulin-receptor kinase activation, and of the mechanisms by which downstream positive and negative signaling proteins are recruited, will hopefully aid in the development of compounds that act specifically to increase the signaling output from the insulin receptor.
Acknowledgements Support is acknowledged from the National Institutes of Health (DK052916, NS053414) and from an Irma T. Hirschl-Monique WeillCaulier Career Scientist Award.
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Structure and Mechanism of the Insulin Receptor Tyrosine Kinase Stevan R. Hubbard Skirball Institute of Biomolecular Medicine and Department of Pharmacology, New York University School of Medicine, New York, New York
Introduction Insulin activates signaling pathways that control cellular metabolism and growth [1]. The actions of this essential hormone are mediated by the insulin receptor [2, 3], a member of the receptor tyrosine kinase (RTK) family of cell surface receptors. The RTK family also includes, among others, the receptors for insulin-like growth factor-1 (IGF1), epidermal growth factor (EGF), fibroblast growth factors (FGFs), platelet-derived growth factors (PDGFs), nerve growth factor (NGF), and vascular endothelial growth factors (VEGFs). A large family of non-receptor tyrosine kinases also exists, which includes Src-family kinases, Abl kinases, Janus kinases (Jaks), Syk and Zap70, and Fak. Receptor and non-receptor tyrosine kinases are critical components of signaling pathways in metazoans, controlling cellular proliferation, differentiation, migration, and metabolism [4]. In contrast to most RTKs, which are single-chain receptors that are activated by ligand-induced oligomerization, the insulin receptor is an 22 heterotetramer (Figure 45.1), with disulfide linkages between the two -chains and between the - and -chains. Insulin binding to the chains induces a structural rearrangement within the receptor, resulting in autophosphorylation of specific tyrosines in the -chain: Tyr972 in the juxtamembrane region, Tyr1158, Tyr1162, and Tyr1163 in the activation loop in the kinase domain, and Tyr1328 and Tyr1334 in the C-terminal region (Figure 45.1). Phosphorylation of Tyr1162 and Tyr1163 (activation loop) stimulates catalytic activity, and phosphorylated Tyr972 (pTyr972; juxtamembrane region), pTyr1158, and pTyr1162 (activation loop) serve as docking sites for downstream signaling proteins. The functional role of tyrosine phosphorylation in the C-terminal region has not been fully established. Handbook of Cell Signaling, Three-Volume Set 2 ed. Copyright © 2010 Elsevier Inc. All rights reserved.
Because the insulin receptor is a transmembrane protein, structural studies to understand signaling mechanisms have largely been confined to either the extracellular region or the cytoplasmic region. Recently, the crystal structure of the disulfide-linked dimeric ectodomain of the insulin receptor was determined [5], which reveals an antiparallel “inverted V” arrangement, and suggests how insulin might cross link the ectodomain by binding to the first L domain of one chain and to the first fibronectin type III domain on the other chain. Crystal structures of the insulin-receptor kinase domain (IRK) in the cytoplasmic portion of the chain have been determined in different states of phosphorylation [6, 7] and for several mutants [8, 9]. IRK shares a similar overall architecture with protein serine/threonine kinases, with an N-terminal lobe (N lobe) comprising a five-stranded anti-parallel -sheet and one -helix, and a larger C lobe which is mainly -helical (Figure 45.1) [10]. ATP binds in the cleft between the two lobes, and the tyrosine-containing segment of a protein substrate binds in the substrate binding groove in the C lobe. A key mechanism by which the insulin receptor and other tyrosine and serine/threonine kinases regulate catalytic activity is through reversible positioning of their activation loops [11]. The activation loop of the insulin receptor, with three autophosphorylation sites (Tyr1158, Tyr1162, and Tyr1163), begins with the protein kinase-conserved 1150DFG motif and ends with tyrosine kinase-conserved Pro1172. In the crystal structure of unphosphorylated (basal) IRK, the activation loop traverses the cleft between the two kinase lobes, with Tyr1162 bound in the active site [6]. In addition to obstructing the substrate binding site, this conformation of the activation loop also obstructs the nucleotide binding site, ensuring that Tyr1162 cannot be phosphorylated in cis. The activation loop is dynamic, however, and upon juxtaposition of the two kinase domains through insulin binding to the 307
308
PART | I Initiation: Extracellular and Membrane Events
α α L domain cys-rich domain
β
L domain β fibronectin type III domains
Extracellular 972 Cytoplasmic 1158 1162 1163 1328 1334
juxtamembrane region tyrosine kinase domain
C-terminal tail
Figure 45.1 Overall architecture of the insulin receptor and structure of the tyrosine kinase domain. The -chains are extracellular and the -chains pass through the plasma membrane. The major sites of tyrosine autophosphorylation in the cytoplasmic domain are numbered according to Ebina et al. [2]. There are three fibronectin type III (FnIII) domains in the extracellular portion of the receptor. FnIII-1 is contained in the -chain, as is the N-terminal portion of FnIII-2. The C-terminal portion of FnIII-2 is contained in the -chain, followed by FnIII-3. Solid lines between the -chains and between the - and -chains represent disulfide linkages. On the right, a ribbon diagram of the structure of tris-phosphorylated IRK is shown [7], with the N lobe colored dark gray and the C lobe colored light gray. AMP-PNP is shown in stick representation, as are the three activation-loop phosphotyrosines and the substrate tyrosine.
ectodomain, phosphorylation of the activation loops ensues in trans, resulting in phosphotyrosine-mediated stabilization of a loop configuration that is optimal for catalysis [7].
Protein recruitment to the activated insulin receptor Overview Upon insulin-stimulated activation of its receptor, several signaling proteins are recruited to the receptor, either for signal propagation or for attenuation. Among the proteins recruited for activation of downstream signaling pathways are several adaptor (non-enzymatic) proteins, including the insulin receptor substrate (IRS) proteins, Shc, and SH2B2/ APS. Negative regulators recruited to the receptor include the adaptor protein Grb14 and the protein tyrosine phosphatase PTP1B. Recent structural studies have elucidated the modes of binding between these proteins and the cytoplasmic domain of the insulin receptor. Phosphotyrosine recruitment sites in RTKs usually reside in polypeptide segments that are N-terminal to the kinase domain (juxtamembrane region), or C-terminal to the kinase domain, or in the kinase insert region (between helices D and E). These polypeptide segments are typically
unstructured (non--helix, non--strand) and can easily adopt an extended conformation, which is required for phosphorylation, and, upon phosphorylation, for binding to Src homology-2 (SH2) domains and phosphotyrosinebinding (PTB) domains [12]. A subset of SH2 domains, those contained in SH2B2 and Grb14, specifically binds to the phosphorylated activation loop of the insulin receptor, which represents an unusual SH2-domain binding target because it is multiply phosphorylated and held in a relatively stable, turn-containing (non-extended) conformation. As detailed below, these SH2 domains possess three important properties: (1) they are dimeric, (2) they recognize two phosphotyrosines in the IRK activation loop, and (3) they are inhibited from binding canonical phosphotyrosine sequences.
Recruitment of SH2B2 to the Insulin Receptor SH2B2 is a member of a family of three adaptor proteins, SH2B1–3, previously referred to as SH2B, APS, and Lnk, respectively [13]. These proteins consist of an N-terminal dimerization motif, a pleckstrin homology (PH) domain, an SH2 domain, and polyproline stretches in the N- and C-terminal regions. Recruitment of SH2B2 to the insulin
Chapter | 45 Structure and Mechanism of the Insulin Receptor Tyrosine Kinase
309
Figure 45.2 Structure of the SH2B2 SH2 domain bound to IRK [15]. In this 2 : 2 complex, IRK is shown with a semi-transparent molecular surface. The phosphotyrosines in the activation loop (pTyr1158/1162/1163) are labeled. The two protomers of the SH2B2 SH2-domain dimer (in the middle of the figure) are shown in ribbon representation.
receptor results in phosphorylation of a C-terminal tyrosine residue in SH2B2, Tyr618, which then serves as a binding site for the tyrosine-kinase binding (TKB) domain of Cbl [14, 15]. This signaling pathway culminates in activation of TC10, a Rho-family GTPase, which is thought to facilitate glucose uptake in adipocytes [1, 16]. The interaction between SH2B2 and the insulin receptor is mediated by the SH2 domain of SH2B2, which, as first shown by yeast two-hybrid studies, interacts with the phosphorylated activation loop of IRK [17, 18]. A crystal structure of the SH2B2 SH2 domain [15] revealed that it possesses a non-canonical architecture, in which the C-terminal half of the domain forms a long -helix rather than two strands (-strands E and F) and a shorter -helix (-helix B) as found in a typical SH2 domain. This structural rearrangement facilitates formation of a novel SH2-domain dimer, which interacts with the activation loops in the two kinase domains of the insulin receptor (Figure 45.2) [15]. Formation of the dimer thwarts binding to canonical phosphotyrosine sequences containing a hydrophobic residue at the 3 position (relative to the phosphotyrosine), and confers specificity for the (turn-containing) phosphorylated IRK activation loop. Rather than coordinating a single phosphotyrosine, each protomer of the SH2B2 SH2 dimer coordinates two phosphotyrosines, the first (pTyr1158) in the canonical (arginine-containing) phosphate binding pocket, and the second (pTyr1162) by two lysines in -strand D (Figure 45.2). Thus, the SH2B2 SH2 dimer is a quad phosphotyrosine-recognition module. Of note, despite a sequence identity of 79 percent between the SH2 domains of SH2B1 and SH2B2, the SH2B1 SH2 domain is monomeric, which switches its binding preference from the IRK activation loop to pTyr813 of Jak2, a conventional SH2-domain ligand with a 3 hydrophobic residue [19].
Recruitment of SH2B2 to the phosphorylated activation loop of the insulin receptor prolongs receptor activation [20], presumably through protection from dephosphorylation by protein tyrosine phosphatases such as PTP1B, and selects for phosphorylation of Tyr618 near the C-terminus of SH2B2, 130 residues C-terminal to the SH2 domain. Although there are numerous tyrosine residues in SH2B2 (68-kDa protein), Tyr618 is the predominant site of phosphorylation by the insulin receptor, despite its non-optimal substrate sequence (non-YXM, where is hydrophobic and X is any residue). Selectivity for Tyr618 is evidently due to the binding of the SH2B2 SH2 dimer to the kinase activation loops, which facilitates entry of Tyr618, near the C-terminus of SH2B2, into the IRK active site.
Recruitment of IRS1 and IRS2 to the Insulin Receptor The IRS proteins are a family of four to six adaptor molecules that possess N-terminal PH and PTB domains, followed by more than 900 residues containing multiple sites of tyrosine and serine/threonine phosphorylation [21]. Knockout studies in mice have shown that IRS1 and IRS2 are essential for organismal development and glucose disposal [21]. Despite a conserved domain architecture, common sites of phosphorylation, and overlapping tissue expression, the phenotypes for the IRS1 and IRS2 knockout mice are distinct. IRS1/ mice are 40 percent smaller than wild-type littermates, and exhibit insulin resistance in peripheral tissues [22, 23]. IRS2/ mice are only slightly smaller (10 percent) than wild-type, but are insulin resistant and develop type 2 diabetes due to insufficient pancreatic -cell function [24].
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Many of the tyrosine phosphorylation sites in the C-terminal region of IRS1/2 reside in a YXM motif, which, when phosphorylated by the insulin receptor (or IGF1 receptor), serves as a recruitment site for the SH2 domain(s) of phosphatidylinositol 3-kinase (PI-3K) [25]. Activation of PI-3K through engagement of tyrosinephosphorylated IRS1/2 is required for glucose uptake into insulin-responsive cells [26]. Other tyrosine phosphorylation sites in IRS1/2 recruit the adaptor protein Grb2 and the protein tyrosine phosphatase SHP2 [21]. IRS1/2 also possess numerous sites of serine/threonine phosphorylation that, in general, negatively regulate tyrosine phosphorylation, either in the course of normal negative feedback or in pathologic insulin resistance [27]. The tandem PH–PTB domains in the N-terminal regions of these adaptor proteins function to recruit IRS1/2 to the insulin receptor for phosphorylation. The PTB domains bind to pTyr972 and adjacent residues (NPXY motif) in the juxtamembrane region of the insulin (and IGF1) receptor [28, 29]. The PH domains are also important for recruitment of IRS1/2 to the receptor [30,31]. Previous yeast twohybrid studies provided evidence for a second (in addition to the PTB domain) insulin receptor-interacting region in IRS2, which was named the kinase regulatory-loop binding (KRLB) region [32, 33] or receptor binding domain-2 (RBD2) [34]. This region of IRS2 interacts with the kinase domain of the insulin receptor (IRK) in a phosphodependent manner [32, 34]. The KRLB region was roughly mapped to residues 591–733 [32, 34], which starts approximately 300 residues C-terminal to the PTB domain. This region has a high proportion of glycine, serine, and proline residues, i.e., it is likely unstructured, and contains three tyrosine residues in a YXM motif. Although the corresponding region in IRS1 contains considerable sequence similarity, including the three YXM sites, it does not stably interact with IRK [33, 34]. Mutagenesis studies in the KRLB region identified two non-YXM tyrosine residues, Tyr624 and Tyr628 (mouse numbering), which are critical for this region of IRS2 to bind to IRK [33]. The molecular basis for the interaction of the IRS2 KRLB region with the insulin receptor was elucidated through co-crystallization of a 15-residue peptide from the KRLB region (containing Tyr624 and Tyr628) with phosphorylated IRK [35]. The structure revealed that this region of IRS2 binds in the active site of IRK with Tyr628 poised for phosphorylation (Figure 45.3). Biochemical experiments demonstrated that Tyr628 is phosphorylated by IRK, but with a Km(ATP) that is very high compared to that of a YXM substrate (1.6 mM vs 40 M, respectively). In addition, the phosphorylated Tyr628 peptide retains significant binding affinity, which translates into poor substrate turn over. As a consequence, this segment of IRS2 inhibits phosphorylation by the insulin receptor of other tyrosine sites in IRS2. A possible functional role of the KRLB region is to suppress tyrosine phosphorylation of IRS2, such that only
PART | I Initiation: Extracellular and Membrane Events
Figure 45.3 Structure of the KRLB region of IRS2 bound to IRK [35]. IRK is shown with a semi-transparent molecular surface. The side chains of the KRLB peptide (N- and C-termini are labeled) are shown in stick representation. Tyr624 and Tyr628 of the KRLB region are labeled, the latter of which is bound in the IRK active site.
metabolic pathways via PI-3 K are stimulated by insulin and not mitogenic pathways via Grb2/Ras; there are 10 potential PI-3 K recruitment sites in IRS2 versus a single Grb2 site.
Recruitment of Grb14 to the Insulin Receptor Among the negative regulators of insulin signaling, Grb14 is a member of the Grb7/10/14 family of adaptor proteins [36]. Grb14/ mice show tissue-specific (muscle and liver) increases in glucose disposal, and patients with type 2 diabetes have been shown to have increased Grb14 mRNA levels in adipose tissue [37]. Grb7/10/14 proteins possess several signaling modules, including a Ras-associating (RA) domain, a PH domain, and a C-terminal SH2 domain, as well as polyproline sequences. In addition, these proteins contain a 45-residue region known as BPS (between PH and SH2) [38] or PIR (phosphorylated insulin receptorinteracting region) [39], which is unique to this adaptor family. Biochemical studies have demonstrated that the BPS region of Grb7/10/14 is capable of directly inhibiting the catalytic activities of the insulin and IGF1 receptors [40, 41], with a potency rank of Grb14Grb10Grb7 [41]. A crystal structure of the Grb14 BPS region in complex with phosphorylated IRK revealed at least one mechanism by which Grb14 inhibits signaling by the insulin receptor [42]. In the structure (Figure 45.4), the N-terminal portion of the BPS region binds in the substrate binding groove in the C lobe of the kinase domain. A leucine (Leu376)
Chapter | 45 Structure and Mechanism of the Insulin Receptor Tyrosine Kinase
is inserted in the active site rather than a tyrosine, and the hydrophobic residues at the 1, 3, and 5 positions relative to Leu376 mimic peptide–substrate binding. Thus, the Grb14 BPS region acts as a pseudosubstrate inhibitor to suppress insulin-receptor signaling. After the pseudosubstrate segment, the Grb14 BPS region adopts a 16-residue -helix whose residues make interactions with the phosphorylated activation loop. These interactions fortify the BPS–IRK interaction and also provide specificity; the BPS region of Grb14/10 only inhibits the insulin and IGF1 receptors. The BPS -helix ends near the kinase N lobe, which positions the C-terminal SH2 domain (25-residue BPS–SH2 linker) for engagement of pTyr1158 and pTyr1162 in the activation loop [42]. The SH2 domain of Grb14/10 is, like the SH2B2 SH2 domain, dimeric, but in this case the individual protomers possess a canonical architecture. Binding of canonical phosphotyrosine sequences (with 3 hydrophobic residues) is deterred in the Grb14/10 SH2 domain, not by dimer formation (as for SH2B2), but by a valine in the BG loop (BG3) rather than a glycine (as in Src and Abl) [43].
Recruitment of PTP1B to the Insulin Receptor PTP1B is a key negative regulator of insulin signaling in vivo, as shown by gene-deletion studies in mice. Despite the ability of PTP1B to dephosphorylate numerous RTKs in vitro and in cells, the PTP1B/ mice are of normal size (i.e., no overgrowth phenotype) and exhibit two apparent phenotypes: a hypersensitivity to insulin, due to prolonged insulin-receptor phosphorylation, and resistance to weight gain on a high-fat diet [44, 45]. The molecular basis for PTP1B’s specificity towards the insulin receptor has not been fully elucidated. A crystal structure of PTP1B with a phosphorylated peptide representing the IRK activation loop revealed that a phosphotyrosine (pTyr1163) following the substrate phosphotyrosine (pTyr1162) is a specificity determinant [46]. Because several other RTKs (e.g., IGF1 receptor, TrkA-C, FGF receptors), which do not appear to be targets of PTP1B in vivo, contain tandem phosphotyrosines in their activation loops, other specificity determinants must exist. A crystal structure of a PTP1B–IRK complex was determined that revealed a novel, non-catalytic mode of interaction between the two proteins, in which PTP1B binds not to a phosphotyrosine in the IRK activation loop, but rather to the opposite side of the kinase domain (Figure 45.5). This interaction was evidently facilitated by crystallization in ammonium sulfate, which effectively competes with the activation-loop phosphotyrosines for binding in the PTP1B active site. Although this interaction might be dismissed as a crystallization artifact, the PTP1B– IRK interface comprises residues that are highly characteristic for these two proteins, including Tyr152 and Tyr153 of
311
Figure 45.4 Structure of the BPS region of Grb14 bound to IRK [35]. IRK is shown with a semi-transparent molecular surface. The BPS region (N- and C-termini are labeled) is shown in ribbon representation. Leu376 (labeled), the pseudosubstrate bound in the IRK active site, is shown in stick representation.
Figure 45.5 Structure of a PTP1B–IRK complex. In this 2 : 2 complex, the IRK dimer is in the center of the figure, and the two PTP1B molecules are on the outside, shown in ribbon representation. The molecular (non-crystallographic) two-fold axis is perpendicular to the page.
PTP1B, which had been previously implicated in the interaction between PTB1B and the insulin receptor [47–49]. Moreover, the highly related IGF1 receptor would be incapable of engaging PTP1B in this manner. Thus, it remains an attractive hypothesis that the interaction observed in the crystal structure represents a recruitment mode of binding to localize PTP1B to the insulin receptor, although biochemical evidence for this hypothesis is still outstanding.
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Prospects An understanding of the signaling mechanisms intrinsic to the insulin receptor is not only of fundamental biochemical interest. The prevalence of non-insulin-dependent diabetes mellitus (NIDDM) in developed countries is increasing at an alarming rate. One therapeutic strategy for NIDDM is to activate or potentiate signaling at the level of the insulin receptor. Indeed, small-molecule activators/potentiators of the insulin receptor that act cytoplasmically have been reported [50, 51]. Knowledge of the structural transitions that underlie insulin-receptor kinase activation, and of the mechanisms by which downstream positive and negative signaling proteins are recruited, will hopefully aid in the development of compounds that act specifically to increase the signaling output from the insulin receptor.
Acknowledgements Support is acknowledged from the National Institutes of Health (DK052916, NS053414) and from an Irma T. Hirschl-Monique WeillCaulier Career Scientist Award.
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