Insulin binding and activation of the insulin receptor
The insulin receptor (IR) is a large, disulphide-linked, glycoprotein that spans the cell membrane with its insulin binding surfaces on the outside of the cell and its tyrosine kinase domains on the inside. IR is a symmetrical homodimer that contains two identical binding pockets, each created by the juxtapositioning of two distinct binding sites involving residues from both IR monomers (IR and IR´). The two binding pockets comprise site 1/site 2´ on one side of IR and site 1´ and site 2 on the opposite side. The current model for IR activation is that two distinct surfaces of insulin engage sequentially with either the site 1/site 2´ binding pocket or the site 1´/site 2 pocket. The formation of a site 1 – insulin - site 2 high-affinity, cross-link involves structural changes in both insulin and IR resulting in the activation of the intracellular tyrosine kinase and the initiation of the phosphorylation cascades that drive insulin signaling. Understanding how insulin binding induces signal transduction requires structures of: (i) insulin and IR in their basal states, (ii) insulin bound to the IR ectodomain, (iii) the activated IR kinase domain and (iv) the domain rearrangements associated with the formation of the high affinity insulin/IR complex that initiates activation of the intracellular kinase.
The structure of unbound insulin
Mature insulin consists of two polypeptide chains (A and B) held together by disulphide bonds. The processes involved in insulin biosynthesis were elucidated by Donald Steiner and colleagues beginning in the late 1960s. They showed that insulin was synthesized as a single chain molecule, proinsulin, with the B-chain at its N-terminus, the A-chain at its C-terminus and a 35 residue connecting segment, the C-peptide, in the middle (Figure 1). The mature two chain molecule is generated from proinsulin by two subtilisin-like convertases that carry out the proteolytic removal of the C-peptide.
Figure 1. Sequence of human insulin precursor. The B- and A-chain segments of the processed protein are shown in cyan and light brown color, respectively, with the proteolytically removed C-peptide in gray. Disulfide bonds are indicated in green, while residues adopting a helical conformation in the mature protein are circled in black. (Click to enlarge)The primary chemical structure of mature insulin was determined by Frederick Sanger and colleagues at Cambridge University in the early 1950s. Insulin was the first protein to have its sequence determined and Sanger was awarded his first Nobel Prize in Chemistry in 1958 for this achievement. The pioneering study showed that the human insulin B-chain consisted of 30 amino acids and the A-chain of 21 amino acids, with the B- and A-chains held together by two disulfide bonds CysB7 to CysA7 and CysB19 to CysA20, with a third intra-chain disulfide bond linking CysA6 to CysA11.
Figure 2. Insulin structure. (A) Tertiary structure of the porcine insulin monomer in its 2 Zn rhombohedral form [PDB entry 4INS]. (B) Structure of the human insulin mutant GlyB24 [PDB entry 1HIT], showing the flexibility of the B-chain C-terminus. For clarity, disulfide bonds are omitted in (B).
The crystal structure of porcine insulin was first reportded by Dorothy Hodgkin (née Crowfoot) and colleagues in 1969 as the 2Zn2+-stabilized hexamer at 2.8 Å resolution. At the same time, and in isolation during the Cultural Revolution in China, the so-called “Beijing Insulin Structure Group” was also working to determine the structure of insulin and published their findings in 1972. The data revealed that the insulin monomer consists of a two-layered sandwich with the B-chain overlaying the A-chain. The B-chain consists of an N-terminal segment (residues B1–B6), a type II β-turn (B7–B10) a central α-helix (B9–B19), a type I β-turn (B20–B23), and a C-terminal β-strand (B24–B28), followed by residues B29 and B30, which are less well ordered (Figure 2). The A-chain consists of an N-terminal α-helix (A1–A8), a non-canonical turn (A9–A11), a second α-helix (A12–A18), and a C-terminal segment (A19–A21).
It is now recognized that human insulin is part of a larger family of sequence-related hormones that comprises insulin, the insulin-like growth factors IGF-I and IGF-II, the relaxin peptides relaxin-1, -2, and -3 and the insulin-like peptides INSL3, INSL4, INSL5, and INSL6. The two IGFs each contain a single polypeptide chain, while each of the remaining members of the family contains a pair of chains resulting from the proteolytic processing of a single-chain precursor. Three-dimensional structures have been determined for these other family members and, as expected from their close sequence relationship, all have the same three-helix tertiary structure and the same disulfide-bonding pattern that was determined originally for human insulin.
Receptor binding surfaces of insulin
Extensive biochemical and structural characterization of the hormone over decades has demonstrated that the surfaces of insulin that bind IR sites 1 and 2 comprise residues predominantly drawn from the insulin dimerization (site 1) and hexamerization (site 2) surfaces. These surfaces have both been shown to have closely related counterparts in the insulinlike growth factors (IGFs) IGF-I and IGF-II.
The structure of the insulin receptor dimer in its basal state
Figure 3. Cartoon of the insulin receptor dimer showing the distribution of domains across the α- and β-chains and the location of the α-β disulphide bonds linking the two chains in each monomer and the two α-α disulphide bonds linking the two monomers in the IR dimer. As discussed in the section Insulin receptor and insulin action the insulin receptor (IR) is a disulphide-linked homodimer with each IR monomer composed of several structural modules commonly found in other proteins. As summarized in Figure 3 these are (from the N-terminus to C-terminus): a leucine-rich repeat domain (L1), a cysteine-rich region (CR), a second leucine-rich repeat domain (L2), and three fibronectin type III domains (FnIII-1, FnIII-2, and FnIII-3), with FnIII-2 containing a large (~120 residues) insert domain (ID). The ID contains the furin cleavage site that yields the α-chain and β-chain of the mature receptor monomer. The intra-cellular C-terminal region of the IR monomer contains the tyrosine kinase catalytic domain, which is flanked by two regulatory regions - the juxtamembrane region and the C-tail.
The structure of the extracellular portion of the human IR (approximately 1800 amino acids) was reported by Colin Ward, Mike Lawrence and colleagues in a landmark paper in Nature in 2006 and represented a major milestone in insulin research. As shown in Figure 4 the structure revealed that the IR adopted a folded-over (inverted “V”) conformation that placed putative ligand binding regions in close juxtaposition. One leg of each monomer consists of the L1–CR–L2 modules. The other leg of each monomer comprises the FnIII-1, FnIII-2, and FnIII-3 domains in an extended, linear arrangement. FnIII-2 contains the ID as an extended loop between the C and C´ strands of FnIII-2.
Figure 4. Structure of the IR before insulin binding. Click to enlarge figure. (A) Three-dimensional crystal structure of the IR ectodomain, showing the inverted “V” conformation with respect to the membrane. The background monomer is shown in molecular surface representation (beige) and the foreground monomer in secondary structure schematic representation (gray) with the constituent domains labeled. The disordered portions of the α-chain components of the insert domain are shown as dashed and their conformation shown here is speculative, with the approximate location of the inter-chain disulfide being indicated by a black line. The relative location of the components that make up the insulin binding site within one leg of the inverted “V” are highlighted and include the surface of the central β-sheet of the L1 domain of one monomer (purple), residues at the junction of FnIII-1 and FnIII-2 domains of the adjacent monomer (light green) and the helix formed by the C-terminal segment of α-chain (brown coil). (B) Detail of the conformation of the αCT segment on the surface the central β-sheet of the L1 domain in the crystal structure of the apo-form of the insulin receptor ectodomain. The figure is based on PDB entry 3LOH; (B) is from Smith et al, used by permission. (click to enlarge)The IR homodimer has a two-fold rotation axis that places the L2 domains of each monomer in contact with the FnIII-1 domain of the alternate monomer at the apex of the inverted “V” and places the L1 domains of the each monomer in contact with the FnIII-2 domains of the alternate monomer around the midpoint of the legs of the inverted “V” (Figure 4). At the base of the structure, the C-termini of the two FnIII-3 domains are poised to extend through the cell membrane to the intra-cellular juxtamembrane, kinase, and C-tail domains of the intact receptor. The FnIII-1 and FnIII-2 domains contain unusually large loops between their C and C´ strands. In the FnIII-1 domains, the large CC′ loop enables formation of the Cys524–Cys524 dimer disulfide bond, while in the FnIII-2 domains an entire ID segment is contained within their respective CC′ loops. The single α−β disulfide bond between Cys647 (near the start of the ID) and Cys860 (at the beginning of the C′E loop of FnIII-3) is visible in the structure. The Cys524 α–α disulfide bond lies in a very weakly ordered region of the polypeptide, whereas the α–α disulfide bond(s) involving Cys862, Cys863, and Cys865 lies in a region of the ID (residues 655–755) that is entirely disordered in the crystal.
In a further refinement of the IR structure residues 693 710 from the C terminal region of the IR α chain, the so called “αCT” segment, were shown to be lying across the central β sheet of each L1 domain. This αCT segment is essential for insulin binding and is known from chemical cross-linking to lie in close proximity to the L1 domain. The key features of the interaction of the 693–710 segment with the surface of the central β-sheet of the L1 domain are shown in Figure 4B. In particular, (i) the side chains of residues Phe701 and Phe705 are packed adjacent to each other in a hydrophobic pocket formed by the side chains of the L1 domain residues Leu62, Phe64, Phe88, Phe89, Tyr91, Val94, Phe96, and Arg118 - these two aromatic residues are conserved in type in IGF-1R (as Tyr688 and Phe692, respectively); (ii) the side chain of Tyr708 is packed approximately parallel to the strands of the L1 central β-sheet, in proximity to the side chains of Arg14, Gln34, Leu36, and Phe88 – again this residue is conserved in type in IGF-1R (as Phe695); (iii) the side chains of the αCT residue pair Glu698/Arg702 lie close to each other and interact with the side chains of the L1 domain residue pair Arg118/Glu120 respectively, with the four side chains forming a charge-compensating cluster; and (iv) the side chain of Leu709 is in hydrophobic interaction with the side chains of Leu37 and Phe64.
A remaining feature of interest is the fact that the αCT segment associated with each L1 domain in the IR dimer is contributed by the alternate monomer to that which contributes the L1 domain. This was first predicted based on (i) the relative locations of the αCT segment and residue 755 from the C-terminal end of the ID and (ii) the knowledge that in the uncleaved pro-receptor, the αCT segment of one monomer would lie across the L1 domain of its partner since, proteolytic cleavage of IR into α and β chains occurs after dimer assembly. This trans arrangement of L1 and αCT has since been confirmed experimentally by complementation analysis and direct chemical crosslinking. The observed association of the L1-β2 sheet and the αCT segment as a tandem structural element thus provides the first view of IR site 1 in its insulin-free form.
Insulin binding surfaces of the insulin receptor
The current model for insulin binding proposes that each monomer in the IR dimer contains two different binding sites, referred to as site 1 and site 2, located on two different regions of the IR monomer. Binding of insulin to the low-affinity site (site 1) on either of the α-subunits is followed by a second binding event between the bound insulin and the second site (site 2) of the other IR α-subunit . Negative co-operativity occurs because high-affinity binding can only occur between the pair of sites (sites 1 and 2') on one side of the IR dimer or the corresponding pair of sites (sites 1' and 2) on the alternate side, i.e. the ligand/receptor bridging can oscillate from one side of the receptor dimer to the other. Two insulin molecules cannot bridge both site 1-site 2' and site 1'-site 2 pairs simultaneously.
The 3D structure of the IR dimer (Figure 4) is consistent with the model of insulin binding described above and allows a description of sites 1 and 2 to be made. Site 1 corresponds to the low-affinity site which controls ligand-binding specificity seen in soluble ectodomain constructs and binds residues predominantly from the surface of insulin involved in the formation of insulin dimers. Site 1 includes contributions from several distinct regions of the receptor: the L1β2 surface; the αCT peptide; and in the case of IGF binding, the CR region. The importance of the L1, αCT and CR regions in low-affinity ligand-binding comes from studies of receptors from patients with defective insulin signalling, site-directed mutagenesis, chimeric receptors and chemical cross-linking (reviewed in refs  and ). Only the L1 and CR domains are important determinants of ligand specificity because swapping the regions that contain the last 16 residues (CT) of the α-chain in IR/IGF1R whole-receptor chimeras or mini-receptors has little effect on ligand-binding. The L1 and CR domains contain the regions of greatest difference when the L1-CR-L2 structures of IR and IGF-1R are compared. These are in the L1 domains around Phe39/Ser35 (IR/IGF-1R numbering) and in module 6 of the CR region where there is no sequence identity between the two receptors and very different electrostatic surface potential.
This alignment of the L1/αCT Site1 against the FNIII-1 and FNIII-2 modules of the monomer not contributing the L1 domain, suggests that the second binding site on IR (site 2´) that interacts with the hexamer surface of insulin corresponds to one or more of the loops at the junction of the FnIII-1 and FNIII-2 domains. Evidence supporting the requirement of this region for high-affinity binding comes from: (i) alanine scanning mutagenesis which reveals a reduction in insulin binding affinity upon individual mutation of residues Lys484, Leu552, and Asp591 (from the FnIII-1 domain) and residues Ile602, Lys616, Asp620, and Pro621 (from the FnIII-2 domain); (ii) a bioinformatic analysis, which reveals a conserved patch of residues in the vicinity of the FnIII-1/FnIII- 2 junction and (iii) studies of whole IR/IGF-1R chimeras and IR/IGF-1R hybrid receptors, which indicate that high-affinity insulin-binding requires insulin-specific components within the region 326-524 on the alternate receptor monomer to that providing the L1 domain.
The structure of the tyrosine kinase domain in its basal state
The crystal structure of the human IR tyrosine kinase (TK) domain in its unphosphorylated (basal) state was reported by Stevan Hubbard and colleagues in 1994. This was the first structure of a tyrosine kinase to be reported, though the structure of a serine kinase (cAMP protein kinase) had been reported earlier. Like the serine kinases, the IR TK is composed of two lobes with a single connection between them (Figure 5A). The N-terminal lobe comprises a twisted β-sheet of five anti-parallel β-strands (β1–β5) and one α-helix (αC). The larger C-terminal lobe comprises eight α-helices (αD, αE, αEF, αF, αG, αH, αI, αJ) and four β-strands (β7, β8, β10, β11). The human IR TK lacks β-strands β6 and β9 present in cAMP protein kinase.
Figure 5. Three-dimensional crystal structure of the insulin receptor tyrosine kinase domain. (A) Inactive form, showing the secondary structure of the N- and C-terminal domains. (B) Interaction between the juxtamembrane residues 978–988 (red tube) and the N-terminal domain of the IR tyrosine kinase in its basal state (transparent pink surface); [PDB entry 1P14]. The N-C direction of the juxtamembrane segment (in red, panel B) is indicated by the red arrow. The image in panel B has been rotated 180° around the Y axis and 90° around the X axis relative to panel A. (Click to enlarge)In the inactivated IR TK, one of the three tyrosines in the activation loop, Tyr1162 (IR-B numbering), is bound in the active site but cannot be phosphorylated (in cis) because part of the A-loop interferes with the ATP binding site and the catalytic residue Asp1150 is improperly positioned to co-ordinate MgATP. Upon activation (see later), auto-phosphorylation of residues Tyr1162, Tyr1158, and Tyr1163 occurs in trans by the kinase domain of the second monomer. Thus, in the basal state, Tyr1162 competes with the neighboring β-chain and other protein substrates for binding to the active site, but is not cis-phosphorylated because of steric constraints that prevent simultaneous binding of Tyr1162 and MgATP.
In 2003 Hubbard’s group described the structure of an extended IR kinase construct showing the molecular details of the interaction between the catalytic domain and the juxtamembrane region (Figure 5B). In IR, the proximal juxtamembrane regions show TK inhibition through the highly conserved residue Tyr984 (IR-B numbering, equivalent to Tyr972 in IR-A). This tyrosine residue interacts with several conserved residues in the N-terminal lobe of the IR kinase domain, stabilizing a non-productive position of the αC helix. This juxtamembrane inhibition in IR is more significant than that contributed by the activation loop since, in the full-length IR found on cells, mutation of Tyr984 to Ala increases the basal phosphorylation state 30-fold, 10 times greater than the three-fold increase seen following mutation of the activation loop residue Tyr1162 to Asp.
The direction of the juxtamembrane segment relative to the lobes of the catalytic domain (Figure 5B) implies that, in the basal state, the catalytic domain is partially wrapped up and held inverted with respect to the cell membrane rather than be exposed and suspended from the end of the 41-residue (~140 Å) strand of juxtamembrane polypeptide (see Insulin receptor and insulin action Figure 4). We suggest that ligand binding results in an as yet unknown domain re-arrangement within the ectodomain that in turn affects the juxtamembrane/catalytic domain interaction, resulting in the release of the sequestered kinase domains and their subsequent transphosphorylation.
The structure of the insulin/insulin receptor complex
The complex with the major binding site - Site1 - In 2013 the Lawrence/Ward group described, for the first time, the structure of insulin bound to Site1, the major binding site on IR, composed of the central β sheet of the L1 domain and αCT. The result (Figure 6) was surprising. Firstly the direct interaction of insulin with the L1 domain of IR is seen to be sparse, the hormone instead engaging predominantly with the αCT segment of the site 1 tandem element. Secondly, the αCT segment is, with respect to its apo-IR counterpart, both displaced on the surface of the L1 β2 sheet, C-terminally extended to include residues 711-715 and N-terminally disordered (residues 697-704).
Figure 6. Structure of the insulin / IR site 1 complex. (a) Both IR monomers are shown in secondary structure schematic representation with the constituent domains labeled. The location of the disordered portions of the α-chain components of the insert domain (ID) shown here is speculative. The relative location of the components that make up the insulin binding site within one leg of the inverted “V” is circled and includes the surface of the central β-sheet of the L1 domain of one monomer (cyan), residues at the junction of FnIII-1 and FnIII-2 domains of the adjacent monomer (bright and dull green) and the helix formed by the C-terminal segment of α-chain (magenta coil). (b) Detail of the insulin - αCT – L1 domain complex (site 1 complex) based on PDB entry 4OGA. It is equivalent to the circled region on the left hand side of panel (a) viewed from the front. The directions of the respective polypeptide chains of insulin and the αCT segment are indicated by the positioning of a small blue sphere at the N-terminus of each chain and a small red sphere at each C-terminus. (c) As in panel (b) viewed from the top looking down the barrel of the L1 domain β-helix.
The αCT helix occupies volume that would otherwise contain insulin residues B26-B30 if the latter retained their receptor-free (storage-form) conformation (Figures 6b and 7). This observation confirms the so-called detachment model of insulin binding in which the C-terminal region of the B-chain displaces to expose the mostly hydrophobic core of the hormone. The detachment model arose originally from a number of observations, including (a) that the single chain B29-A1 peptide linked insulin molecule is completely devoid of biological activity despite retaining the same crystal structure as that of wild type insulin, (b) that the B chain C terminal region (residues B20-B30) of the active PheB24Gly human insulin mutant is detected to be disordered in solution, and (c) that the structure of the insulin ValA3Leu clinical mutant, which exhibits a 500-fold reduction in insulin affinity relative to wild-type, has a structure identical to that of the native hormone and within which the side chain of the variant residue remains buried.
The two most critical hormone-engaging residues in the αCT segment are: (i) His710, which inserts into a pocket formed by invariant insulin residues ValA3, GlyB8, SerB9 and ValB12; and (ii) Phe714, which occupies a hydrophobic crevice formed by invariant insulin residues GlyA1, IleA2, TyrA19, LeuB11, ValB12 and LeuB15.
Frustratingly the insulin B chain C terminal segment that includes residues known to be important in insulin binding (GlyB23, PheB24 and PheB25), was unresolved in the initial insulin/IR site 1 complexes. It is predicted to lie between the αCT segment and the proximal CR domain since different photo-probes attached to insulin LysB29 can label either CR or L1. In addition, such a location must be the case for their counterparts in the single-chain molecules pro-insulin and the IGFs upon their binding to IR or IGF-1R. Further analysis of new data resolved this issue. The results showed that on binding to site 1, the B-chain C-terminal segment detaches from the hormone core by undergoing two rotations that result in its repositioning between αCT and L1 (Figure 6b). The first is rotation of B20-B27 by ~ 10° about B20 and the second a further rotation of B20-B27 by ~ 50° about B24. Although the first rotation effects maximal displacement at PheB24, the latter side chain undergoes a compensating rotameric change preserving contacts with the B-chain helix. The second rotation
Figure 7. Structural changes in insulin on receptor binding. (a) Structure of insulin in its storage form. (b) structure of insulin when bound to IR site 1. (c) an overlay of the two structures. The directions of the A and B polypeptide chains of insulin are indicated by the positioning of a small blue sphere at the N-terminus of each chain and a small red sphere at each C-terminus. positions B25–B27 anti-parallel to the first strand of L1–β2 and nearly perpendicular to the B-chain α-helix (Figure 6b). This series of conformational changes couples detachment of the insulin B-chain C-terminal strand with insertion of the αCT helix (residues 705–714) into the volume occupied by residues B25–B30 in the free hormone. The salient feature of the B24–B27 segment in the IR complex is its alternating pattern of side-chain contacts with L1 and αCT: PheB24 and TyrB26 are primarily directed toward L1 whereas PheB25, and ThrB27 toward αCT. Such alternation was foreshadowed by photo–cross-linking studies of the insulin-holoreceptor complex. A comparison of insulin in its closed (storage form) and open (receptor engaged form) is provided in Figure 7.
The refined structure also provided data for the location of the last four residues (716-719) of the IR α-chain that were disordered in the initial structure. Residues 716–719 are found to be directed away from L1 in an irregular conformation (Figure 6b). Although detailed conformations are uncertain, it is clear that the displaced B24–B26 segment inserts between the first strand of the L1–β2 sheet and αCT residues 715–718 (Figure 6b); contacts with PheB25 (above) stabilize residues 716–719.
In conclusion, the insulin/IR site 1 complex structures provide an explanation for a vast body of biochemical data relating to the insulin/IR interaction and reveal how insulin switches between a closed, stable ‘storage form’ and an open ‘receptor activating’ form. Opening of this hinge enables conserved nonpolar side chains (IleA2, ValA3, ValB12, PheB24, and PheB25) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. These findings rationalize properties of clinical mutations in the insulin family and provide a novel foundation for designing therapeutic analogs.
The structure of the high affinity site 1-Site 2´ complex with insulin- To date no structure is available of insulin cross-linking both IR binding sites, Sites 1 and Site 2´. Nevertheless, the site 1 complexed structures described above support the view that Site 2 most likely lies at the junction of the FnIII-1 and FnIII-2 domains of the IR monomer opposite to that providing the L1 domain to site 1. The structural re-arrangements associated with high affinity insulin binding and activation of the IR tyrosine kinase remain to be established. The existing structural data have highlighted four sites of inter-domain flexibility within IR, notably at the CR-L2, L2-FnIII-1, FnIII-1-FnIII-2 and FnIII-2-FnIII-3 junctions. These would permit the receptor increased capacity to access conformations required for signal induction. The evidence comparing limited versus more extensive domain movements in IR activation following on ligand binding is discussed by Ward et al..
The structure of the activated IR tyrosine kinase
Figure 8. Overlay of the inactive (blue, PDB entry 1IRK) and activated (orange, PDB entry 1IR3) forms of the domain, showing the displacement from the catalytic site of the activation loop (residues 1149–1170, highlighted as a thicker ribbon within each form) and the concomitant domain rotation which are observed upon activation. (Click to enlarge)The structure of the activated phosphorylated IR kinase in complex with a peptide substrate was reported by Stevan Hubbard in 1997. It revealed that auto-phosphorylation of the three tyrosines in the A-loop leads to a dramatic change in its configuration. In the phosphorylated state, the A-loop is displaced by approximately 30 Å, resulting in unrestricted access to the binding site for MgATP and protein substrates. This movement facilitates a functional spatial arrangement of Lys1030 and Glu1047 (IR-B numbering), the residues involved in MgATP coordination, and of Asp1150, which is part of the highly conserved Asp–Phe–Gly triad. The A-loop rearrangement also leads to closure of the N and C-terminal lobes, which is necessary for productive ATP binding. This closure involves significant rotation of the N-terminal lobe (Figure 8).
As discussed earlier, the major inhibition of the kinase domain is by Tyr984 (IR-B numbering) in the juxtamembrane segment. Its interaction with, and inhibition of, the kinase domain would be significantly affected by phosphorylation. The precise way in which insulin binding to the extracellular domains of the IR induces domain re-arrangements that break these intracellular associations and result in kinase activation are not known.
As summarized above there are now structures available for: (a) insulin in its receptor-free form, (b) IR ectodomain in its hormone-free form, (c) the tyrosine kinase domain in its inactive conformation, (d) insulin bound to IR site 1, and (e) the tyrosine kinase domain in its active (phosphorylated) conformation. What remain to be elucidated are the structure of the high-affinity complex wherein insulin cross-links receptor sites 1 and 2′ and induces changes in the relative positions of the subdomains of the extracellular portion of the IR dimer as well as the details on how these ectodomain rearrangements affect the trans-membrane and cytoplasmic regions of the receptor resulting in kinase activation and the initiation of signal transduction. The transmembrane segment of IR (residues 918-940, IR-A numbering) has been analysed in micelles by NMR and the region from Leu923 to Tyr937 shown to adopt a well-defined helical structure with a kink formed by Gly921-Pro922 at the N-terminus. Cross-linking studies suggested that it may form dimers or oligomers under micelle conditions. The idea that in the basal state the TM regions of the IR dimer are themselves in contact is supported by the demonstration that IR can be activated by an exogenously supplied 24 residue peptide (Lys 917-Arg943) containing the complete transmembrane segment. This implies that ligand-induced activation of the IR involves separation of the interacting TM regions of the IR dimer. How the force is generated to pull these interacting segments apart on insulin binding remains to be established.
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