Theinsulin receptor (IR) is atransmembrane receptor that is activated byinsulin,IGF-I,IGF-II and belongs to the large class ofreceptor tyrosine kinase.[5] Metabolically, the insulin receptor plays a key role in the regulation ofglucose homeostasis; a functional process that under degenerate conditions may result in a range of clinical manifestations includingdiabetes andcancer.[6][7] Insulin signalling controls access to blood glucose in body cells. When insulin falls, especially in those with high insulin sensitivity, body cells begin only to have access to lipids that do not require transport across the membrane. So, in this way, insulin is the key regulator of fat metabolism as well. Biochemically, the insulin receptor is encoded by a singlegeneINSR, from whichalternate splicing during transcription results in either IR-A or IR-Bisoforms.[8] Downstream post-translational events of either isoform result in the formation of a proteolytically cleaved α and β subunit, which upon combination are ultimately capable of homo or hetero-dimerisation to produce the ≈320 kDa disulfide-linked transmembrane insulin receptor.[8]
Initially,transcription of alternative splice variants derived from theINSR gene aretranslated to form one of two monomeric isomers; IR-A in whichexon 11 is excluded, and IR-B in which exon 11 is included. Inclusion of exon 11 results in the addition of 12 amino acids upstream of the intrinsicfurin proteolytic cleavage site.
Colour-coded schematic of the insulin receptor
Upon receptor dimerisation, afterproteolytic cleavage into the α- and β-chains, the additional 12 amino acids remain present at theC-terminus of the α-chain (designated αCT) where they are predicted to influence receptor–ligand interaction.[9]
Each isometricmonomer is structurally organized into 8 distinct domains consists of; a leucine-rich repeat domain (L1, residues 1–157), a cysteine-rich region (CR, residues 158–310), an additional leucine rich repeat domain (L2, residues 311–470), threefibronectin type III domains; FnIII-1 (residues 471–595), FnIII-2 (residues 596–808) and FnIII-3 (residues 809–906). Additionally, an insert domain (ID, residues 638–756) resides within FnIII-2, containing the α/β furin cleavage site, from which proteolysis results in both IDα and IDβ domains. Within the β-chain, downstream of the FnIII-3 domain lies a transmembrane helix (TH) and intracellular juxtamembrane (JM) region, just upstream of the intracellular tyrosine kinase (TK) catalytic domain, responsible for subsequent intracellular signaling pathways.[10]
Upon cleavage of the monomer to its respective α- and β-chains, receptor hetero or homo-dimerisation is maintained covalently between chains by a single disulphide link and between monomers in the dimer by two disulphide links extending from each α-chain. The overall 3Dectodomain structure, possessing four ligand binding sites, resembles an inverted 'V', with the each monomer rotated approximately 2-fold about an axis running parallel to the inverted 'V' and L2 and FnIII-1 domains from each monomer forming the inverted 'V's apex.[10][11]
Ligand-induced conformation changes in the full-length human insulin receptor reconstituted in nanodiscs. Left - unactivated receptor conformation; right - insulin-activated receptor conformation. The changes are visualized with the electron microscopy of an individual molecule (upper panel) and schematically depicted as a cartoon (lower panel).[12]Left - cryo-EM structure of the ligand-saturated IR ectodomain; right - 4 binding sites and IR structure upon binding schematically depicted as a cartoon.[13]
The insulin receptor's endogenous ligands includeinsulin,IGF-I andIGF-II. Using acryo-EM, structural insight into conformational changes upon insulin binding was provided. Binding of ligand to the α-chains of the IR dimeric ectodomain shifts it from an inverted V-shape to a T-shaped conformation, and this change is propagated structurally to the transmembrane domains, which get closer, eventually leading to autophosphorylation of various tyrosine residues within the intracellular TK domain of the β-chain.[12] These changes facilitate the recruitment of specificadapter proteins such as the insulin receptor substrate proteins (IRS) in addition toSH2-B (Src Homology 2 - B ),APS and protein phosphatases, such asPTP1B, eventually promoting downstream processes involving blood glucose homeostasis.[14]
Strictly speaking the relationship between IR and ligand shows complex allosteric properties. This was indicated with the use of aScatchard plots which identified that the measurement of the ratio of IR bound ligand to unbound ligand does not follow a linear relationship with respect to changes in the concentration of IR bound ligand, suggesting that the IR and its respective ligand share a relationship ofcooperative binding.[15] Furthermore, the observation that the rate of IR-ligand dissociation is accelerated upon addition of unbound ligand implies that the nature of this cooperation is negative; said differently, that the initial binding of ligand to the IR inhibits further binding to its second active site - exhibition of allosteric inhibition.[15]
These models state that each IR monomer possesses 2 insulin binding sites; site 1, which binds to the 'classical' binding surface ofinsulin: consisting of L1 plus αCT domains and site 2, consisting of loops at the junction of FnIII-1 and FnIII-2 predicted to bind to the 'novel' hexamer face binding site of insulin.[5] As each monomer contributing to the IR ectodomain exhibits 3D 'mirrored' complementarity, N-terminal site 1 of one monomer ultimately faces C-terminal site 2 of the second monomer, where this is also true for each monomers mirrored complement (the opposite side of the ectodomain structure). Current literature distinguishes the complement binding sites by designating the second monomer's site 1 and site 2 nomenclature as either site 3 and site 4 or as site 1' and site 2' respectively.[5][14]As such, these models state that each IR may bind to an insulin molecule (which has two binding surfaces) via 4 locations, being site 1, 2, (3/1') or (4/2'). As each site 1 proximally faces site 2, upon insulin binding to a specific site,'crosslinking' via ligand between monomers is predicted to occur (i.e. as [monomer 1 Site 1 - Insulin - monomer 2 Site (4/2')] or as [monomer 1 Site 2 - Insulin - monomer 2 site (3/1')]). In accordance with current mathematical modelling of IR-insulin kinetics, there are two important consequences to the events of insulin crosslinking; 1. that by the aforementioned observation of negative cooperation between IR and its ligand that subsequent binding of ligand to the IR is reduced and 2. that the physical action of crosslinking brings the ectodomain into such aconformation that is required for intracellular tyrosine phosphorylation events to ensue (i.e. these events serve as the requirements for receptor activation and eventual maintenance of blood glucose homeostasis).[14]
Visualization of full length IR complexes is not yet available due to many constraints. Visualization of full length IR–insulin complexes is not yet available due to flexible link of transmembrane (TM) domains with extracellular domain and intracellular domain. The transmembrane (TM) domains are critical for activation and downstream signaling. Stabilization of TM domains may be result of phosphatidylinositol. Meanwhile, visualization of full length IR–downstream proteins is challenging because of transient nature of association, the phosphorylation receptor requirement, and the unfixed relative orientation.[16]
Applying cryo-EM andmolecular dynamics simulations of receptor reconstituted innanodiscs, the structure of the entire dimeric insulin receptor ectodomain with four insulin molecules bound was visualized, therefore confirming and directly showing biochemically predicted 4 binding locations.[13]
The insulin receptor is a type oftyrosine kinase receptor, in which the binding of an agonistic ligand triggersautophosphorylation of the tyrosine residues, with each subunit phosphorylating its partner. The addition of the phosphate groups generates a binding site for theinsulin receptor substrate (IRS-1), which is subsequently activated via phosphorylation. The activated IRS-1 initiates the signal transduction pathway and binds tophosphoinositide 3-kinase (PI3K), in turn causing its activation. This then catalyses the conversion ofPhosphatidylinositol 4,5-bisphosphate intoPhosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 acts as a secondary messenger and induces the activation of phosphatidylinositol dependent protein kinase, which then activates several other kinases – most notablyprotein kinase B, (PKB, also known as Akt). PKB triggers the translocation of glucose transporter (GLUT4) containing vesicles to the cell membrane, via the activation ofSNARE proteins, to facilitate the diffusion of glucose into the cell. PKB also phosphorylates and inhibitsglycogen synthase kinase, which is an enzyme that inhibitsglycogen synthase. Therefore, PKB acts to start the process of glycogenesis, which ultimately reduces blood-glucose concentration.[18]
Signal transduction of Insulin
Effect of insulin on glucose uptake and metabolism. Insulin binds to its receptor (1), which, in turn, starts many protein activation cascades (2). These include: translocation of Glut-4 transporter to the plasma membrane and influx of glucose (3), glycogen synthesis (4), glycolysis (5), and fatty acid synthesis (6).
Signal transduction of Insulin: At the end of the transduction process, the activated protein binds to thePIP2 phospholipids embedded in the membrane.
The activated IRS-1 acts as a secondary messenger within the cell to stimulate the transcription of insulin-regulated genes. First, the protein Grb2 binds the P-Tyr residue of IRS-1 in itsSH2 domain.Grb2 is then able to bind SOS, which in turn catalyzes the replacement of bound GDP with GTP on Ras, aG protein. This protein then begins a phosphorylation cascade, culminating in the activation of mitogen-activated protein kinase (MAPK), which enters the nucleus and phosphorylates various nuclear transcription factors (such asElk1).
Glycogen synthesis is also stimulated by the insulin receptor via IRS-1. In this case, it is theSH2 domain ofPI-3 kinase (PI-3K) that binds the P-Tyr of IRS-1. Now activated, PI-3K can convert the membrane lipidphosphatidylinositol 4,5-bisphosphate (PIP2) tophosphatidylinositol 3,4,5-triphosphate (PIP3). This indirectly activates a protein kinase, PKB (Akt), via phosphorylation. PKB then phosphorylates several target proteins, includingglycogen synthase kinase 3 (GSK-3). GSK-3 is responsible for phosphorylating (and thus deactivating) glycogen synthase. When GSK-3 is phosphorylated, it is deactivated, and prevented from deactivating glycogen synthase. In this roundabout manner, insulin increases glycogen synthesis.
Once an insulin molecule has docked onto the receptor and effected its action, it may be released back into the extracellular environment or it may be degraded by the cell. Degradation normally involvesendocytosis of the insulin-receptor complex followed by the action ofinsulin degrading enzyme. Most insulin molecules are degraded byliver cells. It has been estimated that a typical insulin molecule is finally degraded about 71 minutes after its initial release into circulation.[19]
Besides the metabolic function, insulin receptors are also expressed on immune cells, such as macrophages, B cells, and T cells. On T cells, the expression of insulin receptors is undetectable during the resting state but up-regulated uponT-cell receptor (TCR) activation. Indeed,insulin has been shown when supplied exogenously to promotein vitro T cell proliferation in animal models. Insulin receptor signalling is important for maximizing the potential effect of T cells during acute infection and inflammation.[20][21]
The main activity of activation of the insulin receptor is inducing glucose uptake. For this reason "insulin insensitivity", or a decrease in insulin receptor signaling, leads todiabetes mellitus type 2 – the cells are unable to take up glucose, and the result ishyperglycemia (an increase in circulating glucose), and all the sequelae that result from diabetes.
A few patients with homozygous mutations in theINSR gene have been described, which causesDonohue syndrome or Leprechaunism. Thisautosomal recessive disorder results in a totally non-functional insulin receptor. These patients have low-set, often protuberant, ears, flared nostrils, thickened lips, and severe growth retardation. In most cases, the outlook for these patients is extremely poor, with death occurring within the first year of life. Other mutations of the same gene cause the less severeRabson-Mendenhall syndrome, in which patients have characteristically abnormal teeth, hypertrophicgingiva (gums), and enlargement of thepineal gland. Both diseases present with fluctuations of theglucose level: After a meal the glucose is initially very high, and then falls rapidly to abnormally low levels.[22] Other genetic mutations to the insulin receptor gene can cause Severe Insulin Resistance.[23]
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