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Feingold KR, Anawalt B, Boyce A, et al., editors. Endotext . Southern Dartmouth (MA): MDText.com, Inc.; 2000-.


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The Insulin Receptor and Its Signal Transduction Network

Pierre De Meyts, MD, PhD, F.A.C.E.

Author Information
Visiting Professor, de Duve Institute, B-1200 Brussels, Belgium; De Meyts R&D Consulting, B-1950 Kraainem, Belgium; worldwide Research outside Affairs, Novo Nordisk A/S, DK-2760 Måløv, Denmark.

ABSTRACT

Insulin is an anabolic peptide hormone secreted through the b cell of the kidneys acting through a receptor located in the membrane the target cells - significant ones gift liver (where it disclosure glucose storage into glycogen and also decreases glucose output), and also skeletal muscle and also fat (where the stimulates glucose transport through translocation the GLUT4), but additionally b cells, brain cells and also in truth most cells, whereby it has pleiotropic effects.

The receptor belongs to the receptor tyrosine kinase superfamily and also has orthologues in every metazoans. The framework of the unbound extracellular domain ("apo-receptor") has been solved. Insulin binding to two unique sites on every a subunit of the receptor, crosslinking the two receptor halves to develop high affinity. The framework of the site 1 interface has additionally been solved, and the framework of the inactive and activated tyrosine kinase, revealing the activation through phosphorylation of an autoinhibitory loop.

The receptor activates a facility intracellular signaling network through IRS proteins and the canonical PI3K and ERK cascades. Overall and also tissue-specific target gene disruption in mice has actually explored the duty of many of the signaling protein in creating the kind 2 diabetes phenotype, through some how amazing results. Insulin signaling in the liver and b cabinet is arising as the major determinant in preventing kind 2 diabetes, with the integrative duty of molecules prefer IRS2 and also FOXO, preventing b cabinet dedifferentiation. The emerging brand-new biology that diabetes opens novel therapeutic avenues for the 442 million type 2 diabetics worldwide. For finish coverage that this and all related areas of Endocrinology, please visit our cost-free on-line web-textbook, www.endotext.org.


INTRODUCTION

Insulin is one anabolic peptide hormone secreted by the b-cells that the pancreas the plays a an important role in the regulation of human being metabolism (Fig. 1) (1). The biosynthesis, secretion, structure and structure-activity relationships space thoroughly the evaluation by Michel Weiss and also colleagues in Endotext (2).

Although insulin is widely regarded as a glucose homeostasis regulating hormone, that is now known to have actually a much more comprehensive pleiotropic role. An insulin-like signaling system exists in every metazoans (1), and regulates evolutionarily conserved processes including reproduction and lifespan (3-7).


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Figure 1


Pleiotropic actions of insulin v the insulin receptor. Insulin with its receptor influence multiple physiological procedures in the organism (left) by boosting (green arrows) or diminish (red arrows) assorted intracellular metabolic pathways (right). Inspired by figure 2-1 of referral 1.


The concept that insulin action by fostering glucose transport throughout the membrane of target cells (rather 보다 acting directly on enzyme of intermediary metabolism of glucose) was established in 1949 by the above experiment the Rachmiel Levine and colleagues (8), who verified that insulin markedly increased the volume of distribution of non-metabolisable galactose in eviscerated nephrectomized dog from 45-47% the body load to 75%, a figure close to that of full body water. Native this finding castle proposed the adhering to working hypothesis: "Insulin acts upon the cell membrane of specific tissues (skeletal muscle, etc.) in together a manner that the transport of hexoses (and perhaps other substances) from the extracellular fluid into the cell is facilitated. The intracellular fate of the hexoses relies upon the availability of metabolic systems for your transformation. In the situation of glucose, dissimilation, glycogen storage, and change to fat are secondarily stimulated by the rapidity that its entry right into the cell".

This major conceptual advance led the method to the id that insulin acts on a certain cell membrane receptor. The receptor was an initial characterized by radioligand binding studies in the early on 70"s (9-12), and by detailed biochemical studies in the early on 80"s that established the glycoprotein nature and subunit framework of the receptor. I have reviewed the early on steps in insulin receptor research in other places (13). Complying with the demonstrate in 1982 by Ora Rosen"s group that a tyrosine kinase to be closely connected with the insulin receptor (14), several groups showed the the insulin receptor itself is a tyrosine kinase, one enzyme that catalyses the transfer of the g phosphate the ATP to tyrosine residual water on protein substrates, the an initial being the receptor itself. The cloning the the insulin receptor cDNA in 1985 by the groups of Axel Ullrich and also Bill Rutter (15, 16) developed that the insulin receptor indeed belongs to the superfamily that receptor tyrosine kinases (RTKs).


THE RECEPTOR TYROSINE KINASE SUPERFAMILY

The RTKs regulate a diverse array of cellular features in multicell organisms, including cell proliferation, survival, differentiation, migration and also metabolism. The human genome encodes 55 RTKs that fall into 19 subfamilies (Fig. 2) based upon which polypeptide ligands castle bind, their sequence homology and also the modular framework of your extracellular domains. For recent reviews that the structure biology of this superfamily see references 17-22. A detailed analysis of the nature of each subfamily is compiled in recommendation 18.

The RTKs are in general single transmembrane polypeptide chains that cross the cell membrane once. The intracellular part of the receptor consists of a tyrosine kinase domain, inactive in the lack of ligand. In the visibility of ligand bound come the extracellular domain, the RTK becomes an activated dimer, in essence an allosteric dimeric enzyme (23). Over there is debate regarding whether the activation is due to ligand-induced dimerization or to ligand stabilization the a pre-existing monomer-dimer equilibrium (21, 23, 24). The insulin receptor subfamily (number 2 in Fig. 2), which comprizes the insulin receptor, the kind 1 IGF receptor (also called IGF-I receptor) which binding insulin-like growth factors I and also II, and the orphan insulin receptor-related receptor (IRRR), is an exemption in the RTK superfamily in the it exists together a covalent disulfide-linked dimer (with a short basal task kinase) in the lack of ligand. This suggests that the activation that a pre-formed dimer is a an ext plausible version for the superfamily. Top top ligand activation the the dimerized RTK, the kinase domains come in contact and also are activated by transphosphorylation, resulting in phosphorylation of details Tyr residual water in the intracellular part of the receptor external the kinase domain. This phosphorylated residues come to be binding sites for signaling companion proteins that contain SH2 (Src homology 2) domains that also become phosphorylated through the kinase or room activated by conformational changes, and also start the intracellular signal transduction cascade.


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The person receptor tyrosine kinase superfamily. This shows the modular framework of the assorted subfamilies of RTKs encoded in the person genome. Note that the AATYK household of 3 kinases is now taken into consideration a serine/threonine kinase (17), which leaves 55 RTKs grouped right into 19 families. The various modules the constitute the architecture of the extracellular and also intracellular domain names are shown below. A detailed description of the framework of this modules deserve to be uncovered in referral 22. From recommendation 22, used with permission.


MODULAR structure OF THE INSULIN RECEPTOR

The insulin receptor has a modular framework (for evaluation see ref. 25) encoded through a gene (located top top chromosome 19) through 22 exons and also 21 introns (26, Fig. 3). The brief exon 11 the encodes a 12-amino mountain sequence is alternatively spliced, bring about two receptor isoforms (A and also B) the differ slightly in affinity for insulin (27-29). The B isoform binding the IGFs with at the very least 100 times lower affinity 보다 insulin, when the A isoform has significantly higher affinity than the B isoform for IGF-I and especially IGF-II (30) and may pat a duty in tumorigenesis. The IGF-I receptor binding IGF-II through a reduced affinity 보다 IGF-I and also insulin v a 500-fold reduced affinity. The receptors are synthesized as solitary chain preproreceptors that room processed by a furin-like proteolytic enzyme, glycosylated, folded and dimerized to yield the mature a2b2 receptor. In cell expressing both insulin and IGF-I receptors, hybrid receptor are developed consisting of one fifty percent of every (31). Your physiological duty is unknown. Compare sequence evaluation of the insulin/IGF-I receptors and also the associated EGF receptor (32) had led Bajaj et al. To indicate (Fig. 3) that the N-terminal half consists the two huge homologous globular domains, L1 and L2, separated by a cysteine-rich region later predicted to consist that a collection of disulfide-linked modules comparable to those found in the tumor-necrosis aspect (TNF) receptor and laminin. The C-terminal half of the receptors was predicted to consist of 3 fibronectin type III (FnIII) domains. The 2nd FnIII domain contains a large insert domain (120 residues) of unknown structure containing the website of cleavage in between a- and also b-subunits. The disulfide bond in between each a- and also b- subunit entails the cysteins C647 and C860. In enhancement there are a-a de defiders bonds at C524 in the FnIII-1 domains and between the triplet C682-C683 and C685 in the insert domain (Fig. 3). The intracellular part of the a-subunit includes the kinase domain flanked by two regulatory regions, a juxtamembrane region involved in docking insulin receptor substrates (IRS) 1-4 and also Shc and also in receptor internalization, and a C-terminal tail. The IGF-I receptor has actually a comparable modular company (33). The recent progress in the X-ray crystallographic structures of totality ectodomains or pieces of the insulin and IGF-I receptor (see below) has largely validated the structure predictions presented in Fig. 3.


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Modular structure of the insulin receptor. Cartoon that the α2β2 structure of the insulin receptor, drawn to scale. Top top the left fifty percent of the receptor, spans the the 22 exon-encoded sequences. Top top the right half, spans of guess protein modules. Module borders mostly correspond to exon boundaries. L1 and L2: huge domains 1 and 2 (leucine-rich repeats); CR: Cys-rich domain. FnIII-1, FnIII-2, FnIII-3: Fibronectin III domains. ID: insert in FnIII-2. TM: transmembrane domain. JM: juxtamembrane domain. TK: tyrosine kinase domain. C: C-terminal tail. Black rectangle close to FnIII-1: major immunogenic region. Orange dots: N-glycosylation sites. Black dots: ligand binding ”hotspots” identified by solitary amino acids site-directed mutagenesis. The two α-subunits are linked by a disulfide bond in between the two Cys 524 in the an initial FnIII domain. One to three of the triplet Cys at 682, 683 and 685 in the insert in ~ the second FnIII domain are additionally involved in α-α disulfide bridges. Over there is a single disulfide bridge between α and also β subunits in between Cys 647 in the insert domain and also Cys 872 (nomenclature that the B isoform) . Exon 11 is highlighted. Adjusted from referral 25.


STRUCTURE that THE UNBOUND INSULIN RECEPTOR ECTODOMAIN (apo-RECEPTOR)

The stepwise progress over the previous 10 years in fixing the framework of the person insulin receptor ectodomain (34-38) has been recently reviewed (39, 40) and also I will emphasis here on the most salient features, based on the most recent higher-resolution (3.3 Å) structure of the ectodomain tied to 4 monoclonal antitoxin (41) Fab pieces (not displayed here), aided by a novel interactive molecular dynamics strategy (38, Fig. 4). The ectodomain homodimer has actually a two-fold symmetric inverted "V" conformation, through the FnIII-1,-2,-3 modules sticking prefer a direct stem from the cabinet membrane and also the L1-CR-L2 module folding end downwards. The L1 and L2 domains are leucine-rich repeat domains. The L1-CR-L2 module that one receptor monomer packs against the FnIII-1,-2,-3 modules the the alternative receptor monomer. Of utmost prestige for insulin binding, the act 16--amino mountain C-terminal helical segment that the insert domain of one monomer packs versus the main b-sheet of the L1 domain that the alternate receptor monomer. This tandem L1/ aCT aspect (36) creates the significant insulin binding site (site 1, see below). This structure does not contain bound insulin.


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Architectural assembly the the unbound insulin receptor ectodomain. This number is based upon the latest higher resolution framework of the apo-receptor in ~ 3.3 Å resolution (38). A. The α subunit. Domains are labeled as in figure 3. FnIII-2α is the α subunit ingredient of the FnIII-2 domain. B. The β subunit. Domain names are labeled as in figure 3. FnIII-2β is the β subunit component of the FnIII-2 domain. C. The αβ monomer. The monomer shows an reverse V-shaped structure. The arrow denotes the site of proteolytic cleavage that the proreceptor. This framework is the A-isoform of the insulin receptor. Twelve amino acids at the finish of the insert domain that the β subunit are absent from the structure. The B-isoform would have actually 12 more amino mountain encoded through exon 11 in ~ the finish of the αCT domain the the α subunit..D. The α2β2 dimer. The arrow indicates the ar of the triplet of de defiders bonds in between Cys 682, 683 and also 685 that the 2 α subunits. E. The tandem insulin binding site 1 make of the αCT helical segment the one α subunit binding in trans to the beta sheet surface of the L1 domain of the second α subunit. The rest of the α subunit insert (ID), i m sorry is not component of the binding site, is also shown for orientation. F. Idem, rotated 90 degrees. Attracted using DSViewerPro indigenous PDB document Model-S1 2, kindly detailed by Mike Lawrence, based on PDB record 4ZXB complemented making use of IMDFF (38).


DEFINITION of THE RECEPTOR BINDING surface ON THE INSULIN and RECEPTOR MOLECULES

The mechanism and also kinetics the insulin binding to its receptor have actually been broadly studied for over 40 years utilizing radioligands (13). Insulin binding is complex and shows an adverse cooperativity, as shown by curvilinear Scatchard plots and acceleration of the dissociation the a pre-bound insulin tracer in one "infinite" dilution in the visibility of cold insulin (42). Just one insulin molecule binding to the receptor dimer through high affinity; additional insulin binding is of reduced affinity, presumably because of ligand-induced asymmetry. IGF-I binding to the IGF-I receptor shows similar properties.

It to be proposed twenty years ago that high-affinity insulin receptor binding outcomes from insulin having two binding web page 1 and also 2 the crosslink 2 binding web page (1 and 2") located on the two different receptor a subunits (43, 44). In stimulate to describe the an unfavorable cooperativity, ns postulated the the 2 a subunits the the receptor should have actually an antiparallel symmetry in order for sites 1 and 2" and 1" and also 2 to be approximated (44), therefore that alternate crosslinking at every subsite pair may take place upon ligand binding, with ligand-induced asymmetry.

Attempts come map the receptor-binding residual water on the insulin molecule day back more than four decades (45-47). Blundell and also colleagues hypothesized that a largely invariant region on the surface of the insulin monomer to be a an excellent candidate because that the receptor-binding region (now referred to as the "classical binding surface" or website 1), including both A-chain residues A1 Gly, A5 Gln, A19 Tyr, A21 Asn and B-chain residual water B24 Phe, B25 Phe, B26 Tyr, B12 Val and B16 Tyr, numerous of i beg your pardon are additionally involved in insulin dimerization. Alanine scanning mutagenesis that the insulin molecule has actually largely shown the role of the previously postulated site 1 residues in binding affinity but additionally identified brand-new residues mapping to the hexamerization surface ar of insulin (site 2): Ser A12, Leu A13, Glu A17, His B10, Glu B13 and also Glu B17 (13,48,49). Ile A10, which had actually not been included in the alanine scan, was also identified as essential for binding affinity by substitution through the homologous IGF residue Ser (50). The main helix that insulin"s B chain shows up to be a significant binding motif and a "hinge" in between sites 1 and 2 (51, 52). An updated mapping that insulin"s binding web page 1 and 2 is shown in Fig. 5 2 binding sites identical to insulin"s web page 1 and also 2 have likewise been established on IGF-I and also II by site-directed mutagenesis (25,, 53), with website 1 prolonging into the C-domain.


Putative to know of website 1 and 2 on the insulin receptor a-subunits has actually been accomplished by a variety of biochemical approaches consisting of chimeric receptors, site-directed mutagenesis and photoaffinity crosslinking (for evaluation see refs. 25 and also 13). Residues connected in site 1 binding affinity were mapped by alanine scanning to the L1 domain and also to the distant aCT domain at the C-terminus that the insert in the a subunit. These locations were sustained by photoaffinity crosslinking experiments (for evaluation see refs. 25 and also 13). The most remarkable an outcome of together expriments was the finding the contiguous residues Phe B24 and Phe B25 that insulin crosslinked to an extremely distant epitopes that the receptor a subunit, B24 come the N-terminal L1 and also B25 come the C-terminal aCT, saying that this two domains somehow came close together in the folded receptor framework (54,55). Complementation experiments showed that this interaction in between L1 and aCT wake up in trans between the two a subunits fairly than in cis in ~ the very same subunit (56), constituting a tandem binding facet that was solved by X-ray crystallography (36). Finally, the vital insulin A3 residue to be shown additionally to crosslink come aCT (57).

Receptor website 2 was mapped by alanine scanning mutagenesis come the loop regions near the junction that the FnIII1-FnIII2 areas of the a subunit (58)..


STRUCTURE that THE website 1 INSULIN-RECEPTOR COMPLEX

Four crystal structures of insulin bound come truncated insulin receptor constructs at 3.9-4.4 Å resolution, in the visibility of exogenous action peptide and also monoclonal antibodies Fab pieces were solved in 2013 (59). The major surprise of the four brand-new structures to be that, contradictory to predictions, insulin is right bound to L1. Most of the residues from the L1 b sheet surface ar that had actually been mapped by alanine scanning room in truth bound to aCT, no insulin (Fig.5). Most of the website 1 residual water of insulin are in intimate call with aCT, except Val B12 and Tyr B16; these last contacts are, however, vital for high affinity. Check out Table 1 that ref. 40 for a detailed summary of insulin residual water contacts with the receptor residues, as well as the impact of alanine scanning the those residual water on binding affinity. Overwhelming evidence supported a vital role of several of the C-terminal residues (B24-B26) that insulin"s B chain in binding affinity, as well as the occurrence of an adverse cooperativity; the 2nd surprise was the the B22 to B30 segment was invisible in the structures. It is clear, however, that if it kept the very same conformation together in aboriginal insulin, there would be a steric clash v aCT, which supports the earlier concept that the B-chain C-terminal have to "detach" to i found it A2 and A3 (60, 61).

Another important conclusion the this examine was that once the L1 domain from the apo receptor is superimposed on the L1 domain from the insulin-bound structure, that is clear that the other receptor fifty percent must undergo a conformational change in order for the insulin molecule come fit between site 1 and the website 2 in the FnIII1-2 region and to protect against a major steric clash (39, 59). The specific nature of this conformational readjust and its relevance to tyrosine kinase activation is tho unresolved.

The last lacking piece the the website 1 facility puzzle dropped into ar in a recent publication (62, the review in 40), report a refinement in among the 2013 frameworks that now reveals the position of insulin B-chain"s C-terminal B20-B27 (the residues B28-B30 are unimportant because that binding). As expected, residues B24-B26 had to turn away through 50 levels from the core of the molecule in order to do room because that the act segment and also to insert itself in between the an initial strand that the L1-b2 sheet and aCT residual water 715-718, sustaining the partial losing model. Opening of this "hinge" permits conserved nonpolar next chains (Ile A2, Val A3, Val B12, Phe B24 and also Phe B25) to connect the receptor. Phe B24 and Tyr B26 are mostly directed toward L1, when Phe B25 and Thr B27 space oriented towards aCT. Contacts with Phe B25 stabilize residues 716-719.

The current structure (Fig. 6) generally supports ahead mappings of site 1 and also 2 top top the insulin molecule (Fig. 5).

The crystal structure of IGF-I bound come a hybrid microreceptor website 1 complex (insulin receptor L1-CR fragment through the IGF-I receptor plot peptide) was additionally recently solved (63), proving congruent come the insulin bound website 1 complicated shown in Fig. 5; interestingly the IGF-I action peptide is threading with the polypeptide loop developed by the C domain the IGF-I.


Structure of the website 1 insulin-receptor complex. Thorough view of insulin"s website 1 and site 2 residual water in the ternary complex between insulin, L1 and αCT that contains insulin"s B-chain C-terminus. The figure shows the structural partnership of insulin"s website 1 (in magenta) and site 2 (in red) residues through the receptor"s αCT and L1 surfaces. Watch table 1 of referral 40 for more detailed description of the contacts. Attracted by Marek Brzozowski using the CCP4MG programme. PDB file: 4OGA. From referral 40, supplied with permission.


QUANTITATIVE analysis OF THE INSULIN-RECEPTOR BINDING PARAMETERS: THE HARMONIC OSCILLATOR MODEL

The complexity of the ligand binding kinetics come the insulin and IGF-I receptors (negative cooperativity and also ligand concentration dependence of the dissociation rate), in the absence of a an accurate understanding of the molecular mechanisms involved, had precluded for plenty of years the advance of a trustworthy mathematical model, although computer programs were easily accessible to approximate "high and low" affinity constants. The different crosslinking design proposed through De Meyts in 1994 (44), together with progress in the structural analysis of the receptors, resulted in a physical plausible, structure-based model, propose by Vladislav Kiselyov et al. In 2009 (64), based upon the concept of a harmonic oscillator. This model postulates that the tetrade of website 1 and also 2 on every a-subunit, disposed together proposed (44) in an antiparallel fashion, spontaneusly oscillate in between the open and also closed conformations. This enables the ligand, bound either to site 1 or website 2, come crosslink the oscillator, creating high affinity and also slowing down the dissociation rate. Alternative crosslinking come the 2nd set of website will result in acceleration that ligand dissociation from the first crosslinked pair. This allows for a simplification of the variety of intermediary states connected (Fig. 7). The system can be explained with just 5 parameters: a1 and a2, the association price constants come receptor website 1 and also 2, d1 and also d2, the particular dissociation rates, and also kcr, a crosslinking consistent that is the mutual of the moment it bring away the oscillator come close. The in its entirety reaction scheme have the right to be explained by 33 differential equations (plus 2 to account because that the little degree of endocytosis and exocytosis in the cells), which have the right to be quickly fitted by computer system software prefer Mathematica. A detailed description of the method is beyond the border of this chapter, yet suffices to say the it allows a robust estimation of the equilibrium and kinetic parameters for both insulin and IGF-I receptor (Table 1), that we have applied to advice the binding properties of a variety of insulin analogues, and also to the kinetic parameters of the two insulin receptor isoforms (65). The take home message here is that the insulin receptor binding system creates a high affinity site with a Kd of around 0.2 nM by crosslinking 2 partial binding sites v Kds of 6.0 and 400 nM (with similar values because that the IGF-I receptor).


STRUCTURE that THE INSULIN RECEPTOR TYROSINE KINASE

The crystal structure of the insulin receptor tyrosine kinase domain was solved by Stevan Hubbard and also colleagues, both in the inactive state (66) and in the caused state (in the presence of a peptide substrate, a stable ATP analogue and also Mg2+ (67) (Fig. 8). The equivalent structures because that the IGF-I receptor kinase were subsequently addressed (68, 69).

Like in every kinases, the architecture features two structurally distinctive lobes, the N-terminal lobe and the C-terminal lobe (Fig. 8), which kind the catalytic site of the kinase where ATP, bivalent cations and the substrate tyrosine residue get together. The 2 lobes are linked by a linker region that develops a hinge which enables the relative movement of the 2 lobes. The structures revealed a novel autoinhibition mechanism (described in Fig. 8) by which an "activation loop" behaves together a pseudosubstrate the blocks the active site in the basal state (closed configuration) and is stabilised in the open position ~ transphosphorylation of three tyrosines. Much more recent data (70) have presented that the activated insulin and also IGF-I receptor kinases are useful dimers, and also that, in addition to the activation loop phosphorylation, one allosteric stabilization take away place entailing an exchange of the juxtamembrane areas proximal come the kinase domain. For evaluation of the structural facets of the insulin receptor and also other kinases, see referrals 19 and 71, 72.


Structure of the inactive and activated insulin receptor tyrosine kinase (with bound ATP analogue AMP-PNP, peptide substrate and also Mg2+). This figure illustrates the autoinhibition mechanism by which Tyr 1162, among the 3 tyrosines that room autophosphorylated in the activation loop (shown in white) in response to insulin (1158, 1162, 1163) is bound in the active site, hydrogen bonded to a conserved Asp 1132 in the catalytic loop (left). Tyr 1162 in result competes v protein substrates before autophosphorylation. In the set off state (right), the activation loop is tris-phosphorylated and also moves the end of the energetic site. Tyr 1163 becomes hydrogen-bonded to a conserved Arg 1155 in the beginning of the activation loop, which stabilizes the repositioned loop. Additionally shown is the peptide substrate v the WMXM motif. From reference 25.


MECHANISM of INSULIN RECEPTOR ACTIVATION by LIGAND BINDING

Despite the substantial recent development in the structures of the extracellular and also kinase domains of the insulin (and to a lesser level IGF-I) receptors, no structure of the complete unliganded and also liganded receptors is available, and therefore we absence the details of the precise mechanism by which extracellular ligand binding outcomes in approximation and also activation the the kinase domains.

The distance in between the membrane insertions that the stems of the extracellular domain of the insulin apo receptor is ~120 Å. This is too far away for the kinase domains to be approximated if lock are hosted close to the stems and also the street is kept in the caused receptor. Ward et al. Have actually speculated (32) that a ligand-induced conformational readjust causes the descent of the kinase domain names (like a yo yo) native a constrained place where they are partly wrapped increase by the juxtamembrane region and lie near the membrane. This release permits the kinases come approximate and also transphosphorylate (Fig. 9A). This model is partially supported by the 3D framework of a juxtamembrane inhibited kinase domain (73).

In comparison with this model, Lee et al. (74) suggested that insulin binding prises personally the transmembrane domain names in a receptor whereby they are organized close with each other in the inactive receptor (Fig. 9B). This is based on the finding that a 24-residue peptide having the IR transmembrane domain succession (but not the IGF-IR sequence) caused the IR tyrosine kinase. There to be no structural research studies to earlier up this claim.

A system quite different from the over that is donate up by convincing structural and biochemical data, to be proposed through Kavran et al (75). Lock concluded the the molecular mechanism for IR/IGF1R activation involves separation of the transmembrane domains enforced by the unliganded extracellular domains, which maintains the receptors in an inhibited state (i.e. Opposing of the system proposed by Lee et al .(74)). Ligand binding relieves the inhibition by disrupting the L1-FnIII2"-3" interaction that stabilizes the transmembrane domain separation, releasing the transmembrane domains to associate, and permitting autophosphorylation the the kinase domains

Finally, Maruyama (24) has actually recently suggest a common mechanism for all dimeric transmembrane receptors whereby ligand binding come the extracellular domain the receptor dimers induces a rotation the transmembrane domains, followed by rearrangement and/or activation that intracellular domain names (the "rotation model", Fig. 9D)


THE INTRACELLULAR INSULIN RECEPTOR SIGNAL TRANSDUCTION NETWORK

Following the activation that the insulin receptor tyrosine kinase through triphosphorylation that its activation loop, the kinase phosphorylates tyrosine residues exterior the kinase domain that the receptor which create binding sites because that signaling protein partner containing SH2 (src-homology 2) domains (76) or PTB (phosphotyrosine- binding) domains. Unlike other RTKs, the insulin (and IGF-I) receptors do not bind signaling protein directly, but instead tie to the phosphorylated juxtamembrane domain residue Tyr 960 (IR--A numbering) a family of large docking proteins referred to as IRS (Insulin receptor substrate)1-6, the first of which was cloned in 1991 (77, 78), as well as the adapter Shc (Src homology 2 domain containing) (79). These form the nucleus for the assembly the a signal transduction bit that is the beginning hub that the miscellaneous intracellular signaling cascades. A thorough inventory of all the signaling proteins connected in insulin signal transduction pathways (Fig. 10) is past the limit of this review. For much more detailed reviews view refs. 1, 80-84.


In order come apprehend the organisation of the complicated insulin signaling network, a valuable concept is the of "critical nodes" (82). The three ideal studied nodes space the insulin receptor/IRS complex, PI3 kinase (PI3K) and also AKT/PKB.

Most insulin effects appear to be mediated with the interaction of IRS-1 and also -2, and also Shc, with the insulin receptor (81, 82, 85). Other docking proteins like CBL, APS, SH2B, GAB1 and-2 and also DOCK1 and-2 have been less broadly studied (81). IRS proteins contain one N-terminal pleckstrin homology (PH) domain (which attaches come the plasma membrane phospholipids) surrounding to a PTB domain that binding to a phosphorylated NPXY (Y 960) motif in the receptor"s juxtamembrane domain. The main and C-terminal parts of the IRS proteins contain up to 20 potential phosphorylation web page that as soon as phosphorylated by the insulin receptor tie to signaling proteins that contain SH2 domains.

The two main pathways of insulin signaling create from the insulin receptor-IRS node room the phosphatidylinositol 3-kinase (PI3K, a lipid kinase)/AKT (also recognized as PKB or protein kinase B) pathway (86,87) and the Raf/Ras/MEK/ MAPK (mitogen triggered protein kinase, additionally known as ERK or extracellular signal regulated kinase) pathway (88). The PI3K pathway is responsible for most metabolic results of insulin, and also is linked exclusively v IRS, while the MAPK pathway emanates indigenous both IRS and also Shc and also is associated in the regulation that gene expression and, in cooperation with the PI3K pathway, in the manage of cell development ("mitogenesis") and differentiation (82).


The PI3K Signaling Pathway

Activation the the PI3K pathway is prompted by the binding of the p85 or p55 regulation subunit of PI3K (an adapter which has 8 isoforms) come IRS1 and-2, leading to activation that the p110 catalytic subunit (which has three isoforms) and generation of phosphatidylinositol-3,4,5-triphosphate (PIP3), which leads to activation that the three isoforms the AKT/PKB by PDK (phosphoinositide-dependent protein kinase) 1 and also -2 (Fig. 10). PDKs tie to PIP3 in the cabinet membrane and also become thereby activated (82, 86, 87).

Four that the critical downstream substrates of AKT/PKB are mTOR, mammalian target that rapamycin, connected in the regulation of protein synthesis (89); GSK3 (glycogen synthase kinase 3), involved in the regulation of glycogen synthesis (90); FoxO (forkhead box-containing protein, O subfamily) warrior factors, specifically FoxO1, affiliated in the regulation that gluconeogenic and adipogenic genes (91) and AS160 (AKT substrate the 160kDa), associated in glucose transfer (92) (Figs.10 and 11). MTOR is a serine/threonine kinase that acts together a nutrient sensor; the is the catalytic subunit of 2 structurally distinctive complexes,mTORC1 and also mTORC2. It stimulates protein synthetic by phosphorylating eukaryotic translation initiation aspect 4E-binding protein 1 (4EBP1) and p70 ribosom protein S6 kinase (p70S6K).

GSK3 is a serine/ threonine protein kinase the inhibits glycogen synthase (but is additionally involved in various other cellular processes); it is inhibited once phosphorylated through AKT/PKB.

FoxO1 is a transcription aspect that translocates come the cell nucleus in the lack of insulin signal, and stimulates the expression of gene such together PEPCK (phosphoenolpyruvate carboxykinase), the an essential enzyme in gluconeogenesis (82), as well as cyclin G2, an patent cyclin that blocks the cell cycle and is inhibited by insulin (93), and also appears come play a key role in insulin (and IGF-I)-induced mitogenesis. FoxO1 is sequestered in the cytoplasm when phosphorylated by AKT. FoxO1 is extremely conserved in development and, under the name daf16, dram a major role in metabolism and longevity in C. Elegans (91).

AS 160 is a 160-kD AKT substrate the plays a vital role in insulin-stimulated glucose carry (1, 92). This is discussed in some detail in the next section.


The MAPK-ERK Signaling Pathway

Grb2 is an adapter protein that binds to IRS and Shc, and also exists in a facility with SOS (son that sevenless), a guanyl nucleotide exchange element that disclosure GDP/GTP exchange top top the tiny G protein p21 ras (94). This consequently activates the cascade that serine/threonine kinases Raf/MEK/ ERK1-2. Phosphorylated ERK 1-2 translocate come the nucleus if the signal is of sufficient duration and also phosphorylates there an array of transcription factors and also mitogen- and stress- set off protein kinases (1, 88).


INSULIN REGULATION the GLUT4 TRANSLOCATION

The prototypical metabolic impact of insulin is the stimulation of glucose move in adipose tissue and also skeletal and cardiac muscle (1, 8, 83). Glucose disposal into muscle is the major component of insulin activity that avoids postprandial hyperglycemia. This is achieved through the translocation by exocytosis the the insulin-sensitive glucose transporter GLUT4 native intracellular vesicles to the plasma membrane, through a system that is still much from fully understood (for review see 95, 96).

GLUT4 is just one of 13 person glucose transporter isoforms (GLUTs) with 12 membrane-spanning domains, and also is extremely expressed in adipose tissue and also skeletal muscle. GLUTs catalyze hexose transport throughout cell membranes with an ATP-independent, facilitative diffusion mechanism. GLUT4 has the distinctive characteristic the a greatly intracellular disposition in the unstimulated state, in storage vesicles dubbed GSVs that space acutely redistributed in the plasma membrane in solution to insulin and other stimuli choose exercise (96).

The major insulin signaling pathway associated in GSVs translocation is the PI3K/PDK1/AKT2 pathway, through phosphorylation the the AS160 substrate (Fig. 11). AS160 is a GTPase-activating protein that when phosphorylated activates little G proteins referred to as RAB that are associated in membrane traficking, by prevent the exchange of GTP for GDP. Atypical Protein kinases C (PKCs) isoforms show up to be also involved downstream the PDK1 but not v AKT.

A parallel signaling pathway (95) in 3T3L1 adipocytes, emanating from caveolae and lipid rafts has been proposed, that requires the phosphorylation that the adaptor protein Cbl by the insulin receptor, and also results in the activation of a small G protein, TC10, a member the the Rho family members which modulates actin structure. This has not been supported by siRNA-mediated gene silencing experiments (96).

Besides insulin, exercise additionally stimulates glucose transport and GLUT4 translocation with an insulin-independent, AMK -dependent device (83, 96).

A much much more detailed description of the device of GLUT4 translocation deserve to be discovered in ref. 83.


Mechanism of insulin-stimulated glucose transport and also GLUT4 translocation. This cartoon illustrates the stimulation that GLUT4 translocation native intracellular storage vesicles (GSVs) to the plasma membrane by the IRS/PDK1/AKT2/ signaling cascade, exhilaration on the GTPase-activating protein AS160 which activates the small G protein RAB. Patent PKCs are additionally involved. From referral 1, offered with permission.


NEGATIVE REGULATION the INSULIN RECEPTOR SIGNALING, SIGNAL TERMINATION

A multitude of mechanisms room in place to attenuate or end the signal induced through insulin, both in ~ the receptor and post-receptor level (82). The insulin receptor and IRS proteins space negatively regulated by ligand-induced downregulation, by tyrosine protein phosphatases and also by serine phosphorylation. Subsequent measures in the protein kinase cascades are likewise modulated by phosphatases. An adverse feedback loops are important in the fine tuning of the network.


Insulin Receptor and IRS protein Downregulation

The train station dependence between insulin receptor concentration in ~ the cabinet membrane and ambient insulin concentration after long-term exposure was an initial demonstrated in vitro in human cultured cell by Gavin et al. In 1974 (97). This was displayed to be as result of clathrin-dependent endocytosis, a mechanism that entails a dileucine motif in the receptor juxtamembrane domain (98). Receptors are then sorted in endosomes for recycling or deterioration in the lysosomes (for a more detailed evaluation of the instrument involved, see ref. 99). This regulation mechanism has subsequently been extended to the whole class of RTKs (100). In a solution perspective, receptor endocytosis is viewed as a major negative feedback circuit exhilaration on the intake signaling great (101).

Insulin internalization is yet not simply a system of signal termination due to the fact that it has actually been displayed that signal transduction proceeds from the internalized receptor until stopped by phosphatases (99), and also it may be crucial for Shc activation of the Ras-ERK cascade (102, see referral 104 because that review).

Hyperinsulinemia also decreases the expression the IRS1 and also IRS2 in both cultured cells and also in the organization of mice, which has been linked to insulin resistance in animal models (82). This is early out both come increased degradation (by ubiquitination) and decreased synthetic (82).


Serine phosphorylation that IRS Proteins

IRS1 has actually over 70 potential serine phosphorylation sites, the phosphorylation of i m sorry in basic seems to negatively regulate IRS signaling (82, 104). Plenty of of the IRS kinases such together ERK, S6 kinase and c-Jun-N-terminal kinase (JNK) space activated through insulin, arguing that IRS serine phosphorylation is a an unfavorable feedback mechanism in the insulin signaling network (82). The TNFa receptor, involved primarily in apoptosis and inflammation, cause the serine phosphorylation of IRS1 through JNK (82, Fig. 10) and induces insulin resistance in humans, animals and also in vitro (105).


Dephosphorylation of Insulin Signaling proteins by Protein Phosphatases and Phospholipid Phosphatases

Constitutively active phosphatases induce a major attenuation that signals within the processing core of RTK signaling networks (101).

Vanadate and related link inhibit protein tyrosine phosphatases and also increase the tyrosyl phosphorylation the the insulin receptor and also intracellular substrates, and act as insulin mimetics (106).

The major and most studied protein tyrosine phosphatase exhilaration on the insulin receptor is PTP1B. It stays in the endoplasmic reticulum and dephosphorylates the insulin receptor throughout internalization and recycling come the plasma membrane (107, 108). Mice doing not have PTP1B show increased insulin sensitivity and also improved glucose yongin (109, 110).

The dephosphorylation the downstream substrates is excellent by serine/threonine phosphatases (111) and also in the instance of ERK/MAPK by double specificity phosphatases (112).

The task of the PI3K pathway is additionally negatively regulated at the level the PIP3 by phospholipid phosphatases such together the phosphatase and tensin homologue (PTEN, a tumour suppressor) and SH2-containing inositol 5"-phosphatase-2 (SHIP2), which dephosphorylate and also inactivate PIP3. PTEN dephosphorylates phosphoinositides top top the 3"-position conversely, SHIP2 does the on the 5"-position (82, 113).


Other attenuators of insulin receptor signaling: suppressor that cytokine signaling (SOCS) proteins, growth-factor-receptor-bound protein 10/14 (Grb10/14) and plasma-cell membrane glycoprotein-1 (PC-1)

Insulin receptor signaling is also attenuated by suppressor of cytokine signaling (SOCS) proteins, which are mediators that cytokine receptor signaling (like leptin and also IL-6 receptors) v Janus kinases (JAK) and also signal transduction and also activation of warrior (STAT) proteins (114, 115, Fig 10). SOCS 1, -3, -6 and -7 disrupt insulin signaling with binding to the insulin receptor and/or by targeting IRS-1 and IRS-2 for proteasomal degradation (115).

The adaptors Grb10/Grb14, which bind to the insulin (and IGF-I) receptor kinase activation loop, additionally attenuate receptor signaling (116). Interesting way the related adaptors SH2B1/B2 have the opposite effect (116).

Finally, insulin receptor signaling is inhibited by PC-1, also known as the enzyme ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1), and also this was displayed to it is in dependent ~ above its enzymatic activity (117).


Negative Regulation of PI3K and also Insulin activity by the PI3K regulatory Subunit

In enhancement to its function as a positive regulator the PI3K function, the regulatory subunit paradoxically likewise has a function as a an adverse regulator since knockouts of this subunit can improve insulin sensitivity. The putative instrument are questioned in ref.83.


CLINICAL implications OF THE INSULIN RECEPTOR and SIGNAL TRANSDUCTION NETWORK

Type 2 diabetes has actually in recent years got to epidemic proportions, v a full number freshly revised upwards by who to 422 million kind 2 diabetic adults worldwide, pushed by the excessive weight epidemics, surpassing the most downhearted earlier predictions.

Type 2 diabetes is a facility and heterogeneous syndrome, v combinatorial effects of various genetic determinants ("diabetogenes") and lifestyle and also environmental factors (118, 119). It combine alterations in in its entirety insulin sensitivity (the so-called insulin resistance) and also alterations in beta cabinet production and secretion the insulin in response to elevated glucose. There is no consensus as to the relative impact of both defects in "causing" type 2 diabetes, nor in the an accurate succession the molecular events at the tissue and also cellular level bring about the breakthrough of full-blown diabetes.

With the considerable progress in unravelling the insulin receptor signaling network in recent years, numerous studies have addressed the possible dysregulation of this network in resulting in insulin resistance both in vivo and in vitro, in cabinet lines, animal models as well as in humans. In particular, gene invalidation and transgenic approaches in mice have been used extensively to explore the duty of individual football player in the signaling network, alone or in combination, in the maintain of regular insulin sensitivity, as well as, because that some, in common beta cell function. It is fair to say the although v these research studies we have actually learned a lot about the function of individual signaling pathways and molecules in cell role and as whole metabolic regulation in rodents, a clear photo has not emerged regarding what molecule defect(s) precisely causes insulin resistance in human obese and kind 2 diabetic patients, nor as to what is the relative influence of insulin resistance versus beta cell dysfunction in humans. I will therefore not attempt right here to administer an encyclopedic summary of the considerable gene knockout and also transgenic studies, yet only tension a couple of salient and intriguing features; watch ref. 120 for a much more detailed testimonial of early studies and also the excellent and also detailed up-to-date thing by Morris White (83).

The importance of the insulin receptor in metabolic homeostasis was clearly demonstrated by the recognize of assorted compound heterozygous insulin receptor mutations (about 60 so far) in human being syndromes linked with excessive insulin resistance: form A insulin resistance v acanthosis nigricans, leprechaunism, Rabson Mendenhall syndrome (121) and in two siblings v congenital fiber-type dysproportion myopathy (122). Depending upon the place of the mutation in the receptor structure, the functional results have to be ranked in 5 categories (121): 1) impaired synthesis, 2) impaired move to the plasma membrane, 3) impaired insulin binding, 4) impaired transmembrane signaling and also 5) impaired endocytosis, recycling and also degradation.

The finish knockout the the 2 alleles the the insulin receptor in mice led to significant metabolic alterations shortly after suckling. They to be born v moderate growth special needs (-5%). They arisen a significant postnatal growth retardation and skeletal muscle hypotrophy and also died in ~ a main after birth of acute diabetic ketoacidosis (120, 123, 124).

Reported cases of human patients v homozygous insulin receptor deletion room exceptional, likely due to embryonic lethality. Interestingly though, the four instances reported over two decades ago (125-128) proved a milder phenotype (leprechaunism) than the mice dual knockouts and lived numerous months come a year after birth like many leprechauns, showing that in people life is compatible with lack of insulin receptors. This points come the truth that one needs to be mindful in extrapolating findings through rodent hereditary manipulations come conclusions concerning pathophysiology in humans.

Following the demonstrate in cell culture of insulin receptor downregulation in the existence of boosted insulin concentration in the society medium in vitro in 1974 (97), studies verified that the insulin receptor was likewise downregulated in vivo in hyperinsulinemic resistant claims in pet models (ob/hob mice, db/db mice, mouse treated with gold thioglucose, or the Koletsky rat) (129 - 131) and also in turn monocytes (132) or adipocytes (133) the obese humans, back some person studies dissented (134, 135). This caused the concept that receptor downregulation in vivo was a significant determinant of insulin resistance. However, it to be later displayed that heterozygous null mutants (IR +/-) because that the insulin receptor that had only fifty percent the normal match of receptor did not present with any major metabolic abnormalities and also had typical glucose tolerance complying with an intraperitoneal glucose tolerance test, and also had typical insulin levels (123), strongly arguing that downregulation the the insulin receptor alone is not sufficient to cause insulin resistance, and that postreceptor defects may be required, as had actually been previously said (136).

Obvious candidates for a postreceptor defect in insulin resistance room the IRS proteins, considering the critical role the this node in both insulin and IGF-I signal transduction (82). Defects in muscle IRS1 expression and function have to be reported in insulin-resistant states such as weight problems and form 2 diabetes (reviewed in 137). Surprisingly, the homozygous deletion that IRS1 in mice by two various groups (138, 139) induced a rather moderate metabolic phenotype v no type 2 diabetes, regular fasting glycemia, and also normal glucose tolerance in one situation (138) but far-ranging hyperglycemia ~ intraperitoneal glucose fill in the various other (139). A mild level of insulin (and IGF-I) resistance was found in both studies. Both showed marked intra-uterine and also postnatal development retardation.

In contrast, mice v homologous deletion that IRS2 (140) were 10% smaller and mildly insulin resistant at birth, but occurred a significant glucose intolerance and had full-blown diabetes at 10 weeks through both marked insulin resistance and beta cell deficiency.(more than two-fold diminish in beta cabinet mass as early as 4 weeks) with diminished insulin secretion in solution to glucose. These and further data demonstrated that IRS2 dram a crucial integrative function in pancreatic beta cell plasticity and duty (for evaluation see referrals 1, 83), and also support the principle that the beta cell is not just the resource of insulin, but vital target of its actions (141). Tissue-specific knockout the the insulin receptor gene in the b cell led to a defect in their early secretory response similar to the in kind 2 diabetes (142).

Among the numerous full or tissue-specific knockouts the insulin pathway components, i will just stress a few of the unexpected results that tested our current wisdom about diabetes pathophysiology:

As currently mentioned, the target disruption the the p85 regulation subunit that PI3K paradoxically caused hypoglycemia because of increased sensitivity and increased basal and insulin-stimulated glucose disposal in peripheral tissues v a preferential localization the GLUT4 in the plasma membrane (143, 144, see referral 82 for review).

Surprisingly, homozygous deletion of GLUT4 did not create a diabetic phenotype (145), Females had normal glucose levels in fasted and also fed state while males had a 34% reduced fasting glucose and 20% increase in fed glycemia. Both sexes were insulin resistant and hyperinsulinemic, showed development retardation and also had disturbed lipid metabolism. They had actually a shorter life span attached to significant cardiac hypertrophy.

70% of insulin-induced glucose handle goes to skeletal muscle. Tissue-specific knockout the the insulin receptor in skeleton muscle (MIRKO, 146) result as supposed in a significant reduction that insulin-stimulated glucose carry in bones muscle. Surprisingly, the pets showed no hyperglycemia, hyperinsulinemia or evidence for any type of impairment of glucose homeostasis. They had disturbed lipid metabolism.

Also rather counter-intuitively, mice through tissue-specific insulin receptor knockout in adipose tissue (FIRKO, 147) confirmed a markedly decreased fat mass and whole-body triglyceride stores and also were safeguarded from yellow thioglucose-induced and also age associated obesity and also from the obesity connected glucose intolerance.

Finally, the third significant target organization for insulin, the liver, was also targeted by tissue-specific knockout that the insulin receptor (148). The liver accounts because that 30% the the handle of an oral glucose pack through glucose absorb (insulin independent) and also storage right into glycogen, and also accounts because that the significant part that insulin clearance through receptor-mediated endocytosis. The knockout mice verified severe primary insulin resistance and also a defect in insulin clearance. Hence isolated liver insulin resistance is adequate to cause severe defects in glucose homeostasis. The mice additionally developed alterations in liver role and moving morphology.

I will certainly not review right here the comprehensive work excellent in knocking out the insulin receptor in non-classical target tissues (the beta cell has actually been questioned above), except to mention the an essential finding based upon neuron-specific knockout mice the the insulin receptor in the brain (the visibility of which was reported years previously (149.150)) plays crucial role in the control of body weight and also reproduction (151).

Thus from every these studies, the hepatocyte and the b cell have emerged as the major insulin-responsive cells where loss that insulin signaling cause the significant phenotypic alterations in glucose homeostasis discovered in form 2 diabetes.

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Major initiatives have been dedicated in current years by huge consortia to pick potential human being diabetogenes in human type 2 diabetes through genome-wide combination scans (GWAS). So far at least 70 developed susceptibility loci have actually been identified that account because that at many 10% of the familial aggregation, many of i beg your pardon pointing to b cabinet dysfunction quite than insulin resistance (for review see referral 152). No smoking cigarettes gun has been recognize in insulin signalling with the recent exemption of Grb14 discussed over as a negative regulator of insulin signaling (153).