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Insulin Receptor
ContentsFunction of the ReceptorThe insulin receptor is a transmembrane receptor that resides in the plasma membrane and is activated by the binding of insulin.[1] The insulin receptor belongs to the large class of receptor tyrosine kinase (RTKs). RTKs are found at the cell surface and have a high affinity for a particular ligand. RTKs are made up of three distinct parts: an extracellular domain with ligand binding sites, a transmembrane region, and an intracellular domain with the tyrosine kinases that initiate intracellular signaling cascades.[1] The insulin receptor binds the insulin hormone and initiates a cascade of events within the cell. The receptor resides within the plasma membrane of insulin targeted cells. These cells are found in various organs, such as the liver, and tissues, including skeletal muscle and adipose [2]. The insulin receptor is activated by multiple insulin molecules binding to various sites on the receptor.[3] Once activated, the receptor serves as the gateway for the regulation of various cellular processes including glucose transport, glycogen storage, autophagy, apoptosis, and gene expression. Additionally, problems with the insulin receptor are associated with the development of diseases such as Alzheimer's, type II diabetes, and cancer.[4] Through recent cryo-EM structures of the insulin receptor bound in various conformations, a complete three-dimensional understanding of the conformational changes in the insulin receptor upon insulin binding are finally coming into focus. Evaluation of the structural composition and the biochemical properties of the insulin receptor reveals details about the role of the receptor in crucial cellular processes. This page focuses specifically on the insulin receptor's role in glucose homeostasis. InsulinInsulin is a hormone made of two separate amino acid chains that are bound by multiple disulfide bonds. Insulin is synthesized and secreted from the islets of Langerhans of the pancreas in response to high concentrations of glucose in the blood. Once it is secreted, insulin moves through the bloodstream and binds to unactivated insulin receptors residing in the plasma membrane. Binding of insulin to the insulin receptor is a complex process, which involves negative cooperativity among insulin molecules.[3] [5] [1] Current hypotheses propose that the receptor is fully activated only after multiple insulin molecules are bound.[3] The binding of the increased amount of insulin to the insulin receptors will activate their downstream pathways to initiate glucose uptake by the phosphorylation of the Insulin Receptor Substrate (IRS).[6] The transport of extracellular glucose into the cell allows it to be converted to glycogen for storage and later usage. Insulin Receptor Structural OverviewThe insulin receptor is a dimer of heterodimers made of two alpha subunits and two beta subunits. [7] Within the extracellular ectodomain, there are four potential binding sites that can interact with insulin ligands on the extracellular side of the membrane. The full extracellular and intracellular components of the insulin receptor have only been imaged in separate sections but a larger picture of how these sections combine to initiate downstream tyrosine autophosphorylation is emerging.[8] Alpha Subunits The alpha subunits make up the extracellular domain (ectodomain) of the insulin receptor and are the sites of insulin binding. The alpha subunit is comprised of two Leucine rich domains (L1 & L2), a Cysteine rich domain (CR), and a C-Terminal alpha helix (Figure 1). [4] The CT-alpha helix is unique in its position that allows it to reach across the receptor and interact with the insulin at the binding site on the opposing side of the receptor. The alpha subunits are held together by a disulfide bond between cysteine residues at the CYS524 position on each alpha subunit. The disulfide bonds are important to the overall stabilization of the molecule as it binds to insulin. Two types of insulin binding sites are present in the alpha subunits, sites 1 and 1' and sites 2 and 2' (Figure 2). The sites are in pairs because of the heterodimeric nature of the receptor. Due to structural differences, as well as greater surface area and accessibility, binding sites 1 and 1' have much higher affinity than that of sites 2 and 2'. Insulin can also bind at sites 2 and 2', but the location on the back of the beta sheet of the FnIII-1 domain and lack of surface area decreases the likelihood of their binding site becoming occupied as quickly. [3] Cryo-EM has imaged insulin bound structures that displayed a T-shape conformation in the alpha subunits, which make up the receptors extracellular domain region.[3]  Beta SubunitsThe beta subunits spans from the extracellular domain across the transmembrane region and into the intracellular portion of the insulin receptor. The beta subunit is composed of part of fibronectin domain III-2 and all of Fibronectin domain III-3. [4] The beta subunit's FnIII-3 domain has links through the transmembrane region into the intracellular part of the membrane. Cryo-EM provided clear representations of the FnIII-2 and FnIII-3 domains (Figure 1) but are missing the transmembrane and intracellular regions. Although the FnIII-3 domain is connected to the transmembrane and intracellular regions, the active T-shape conformation (Figure 3) likely extends all the way to the tyrosine kinase domain region (see PDB 4XLV).[9] Subunit OrganizationThe alpha and beta subunits of the extracellular domains fold over one another and form a "V" shape when the insulin receptor is inactivated. Upon activation, the extracellular domain undergoes a conformational change and forms a "T" shape. An additional component to the ectodomain is the ''alpha'' chain C-terminal helix, which is also referred to as the "alpha-CT".[3] Each of the dimers has an "alpha"-CT. The alpha-CT is a single alpha-helix and it plays an important role in insulin binding and stabilization of the "T" shape activated conformation. The alpha-CT interacts with a leucine-rich region of the alpha subunit and a fibronectin type III region of the beta subunit to form the insulin binding sites known as site 1 and site 1'.[3] The structure of the extracellular domain is stabilized through multiple disulfide bonds. The alpha subunits are linked through two disulfide bonds, with the main one being between Cys524 of two adjacent alpha subuntis [5]. Cys683 of both alpha subunits are also held together with a disulfide bond.[10] The alpha subunit is also attached to the beta subunit by a disulfide bond between the Cys647 of the alpha subunit and Cys872 of the beta subunit.[10]
FunctionInsulin BindingThe insulin receptor unit has four separate sites for the insulin binding. There are two pairs of two identical binding sites referred to as sites 1 and 1' and sites 2 and 2'. The insulin molecules bind to these sites mostly through hydrophobic interactions, with some of the most crucial residues at sites 1 and 1' being between Cys A7, Cys B7, and His B5 of insulin and Pro495, Phe497, and Arg498 of the insulin receptor FnIII-1 domain.[3] Despite some of the residues included being charged they can still interact hydrophobically in this binding site. For example, due to arginine carrying its positive charge at the end of the side chain, the side chain is bent to allow the hydrophobic part of the side chain to interact with the other hydrophobic residues. The alpha subunits also have significant disulfide linkages that help maintain a compact binging site. At sites 2 and 2', the major residues contributing to these hydrophobic interactions are the Leu 486, Leu 552, and Pro537 of the insulin receptor and Leu A13, Try A14, Leu A16, Leu B6, Ala B14, Leu B17 and Val B18 of the insulin molecule.[3] While the majority of the binding interactions appear similar, sites 1 and 1' have a higher binding affinity than sites 2 and 2' due to site 1 having a larger surface area (706 Å2) exposed for insulin to bind to compared to site 2 (394 Å2).[3] The binding interactions of the insulin molecules in sites 1 and 1' are facilitated by hydrophobic residues of an alpha-helix of the insulin receptor. The insulin molecules in sites 2 and 2' primarily interact with the residues that comprise some of the beta-sheets of the insulin receptor.  At binding sites 1 and 1', a tripartite interaction occurs between three critical parts of the alpha subunits of the insulin receptor.[3] The entire interface of the tripartite interaction involves many residues that are involved with intra-protomer ionic and hydrogen bonding at the binding site. The α-CT chain and the FnIII-1 domain region become in close proximity during the conformational change of the insulin receptor and their interaction involves the following residues: ASP496, ARG498, and ASP499 on the FnIII-1 domain and the LYS703, GLU706, and ASP707 on the α-CT domain. This duo then interacts with the L1 region, specifically ARG14, creating an ideal binding site for the insulin ligand. The FnIII-1 and α-CT are interacting from the two different alpha subunits, which displays a "cross linking" scenario where the domains of the heterodimer can intertwine with each other. The tripartite interaction between the α-CT chain, FnIII-1 domain, and the L1 region is important because it allows for a strong interaction between two subunits of the insulin receptor that maintains and stabilizes the T-shape activation state for the rest of the downstream signaling to occur.[3] Recent studies have demonstrated that at least three insulin molecules have to bind to the insulin receptor to induce the active "T" shape conformation, as binding of two insulin molecules is insufficient to induce a full conformational change.[3] However, this conclusion has not yet been widely confirmed.[3] It has been speculated that activation of the insulin receptor can change based on the concentration of insulin. In low concentrations of insulin, the insulin receptor may not require binding of three insulin molecules in order to exhibit activation. Rather, the level of activity will change in accordance to the availability of insulin.[3] When higher concentrations of insulin are present, the conformational difference between the two-insulin-bound state and the three-insulin-bound state is drastic as the insulin receptor transitions from the inactive "V" shape to the active "T" shape.[3] However, in conditions of low insulin availability, the two-insulin-bound state may be enough to induce partial activation of the receptor.[3] Conformational ChangesThe conformational change between the inverted, inactive "V" shape and the active "T" shape of the insulin receptor is induced by insulin binding. The T shape conformation is well observed in the alpha subunit. It is horizontally composed of L1, CR (including the α-CT chain), and L2 domains and vertically composed of the FnIII-1, 2, and 3 domains (Figure 1). The proper conformational change of the ectodomain of the insulin receptor is crucial for transmitting the signal into the cell. The movements extracellularly cause the two receptor tyrosine kinase domains intracellularly to become close enough to each other to autophosphorylate.[2] This autophosphorylation leads enzymes to become activated in the cell that carries out processes related to insulin signaling such as metabolism and growth. [2]  When an insulin molecule binds to site 1 of the alpha subunit, the respective protomer is recruited and a slight inward movement of the Fibronectin type III domains of the beta subunit is initiated. This is accomplished by the formation of several salt bridges, specifically between Arg498 and Asp499 of the FnIII-1 and Lys703, Glu706, and Asp707 of the alpha-CT.[3] Binding of insulin to both protomers establishes a full activation of the insulin receptor. This activation is demonstrated through the inward movement of both protomers. This motion has been referred to as a "hinge" motion as both protomers "swing" in towards one another.[3] Figure 4 depicts the conformational change and "hinge motion" between the inactive and active forms of an insulin receptor protomer. Upon insulin binding, the beta subunits of the inactive form, shown in blue, are "swung" inward to the active form, shown in orange. When the receptor is in an inverted V shape, the FnIII-3 domains are separated by about 120Å. [11] This distance prevents the initiation of autophosphorylation and downstream signaling by the tyrosine kinase domains on the intracellular side of the receptor. Upon the binding of insulin to multiple binding sites, the conformation change will begin and bring the FnIII-3 domains within 40Å of each other to induce the T shape conformation.[12] [3] As the fibronectin type III domains of the beta subunit swing inward, the alpha subunits also undergo a conformational change upon insulin binding. As insulin binds to site 1, the leucine-rich region of one protomer interacts with the alpha-CT and the FNIII-1 domains of the other protomer to form the tripartite interface binding site.[3] For the tripartite interface to form, the alpha subunits of each protomer must undergo a "folding" motion. While there is an explanation for which conformational changes of the insulin receptor take place, there is no full explanation for the exact mechanism by which the conformational changes are executed in the receptor.[3] Signaling DynamicsThe conformation change from the inactive "V" shape to active T shape state upon insulin binding has a time constant of six minutes (Figure 4) [7] Autophosphorylation of the beta subunits reaches a maximal level in about one minute, and lasts for approximately six to ten minutes. One insulin receptor substrate has a half-life of 3.5 minutes where it is able to be phosphorylated by the tyrosine kinases of the beta subunits and then act as a central hub for further downstream signaling pathways. Once insulin binds to the alpha subunits, the receptor remains active for approximately ten minutes before the insulin is degraded and the receptor returns to its inactive conformation. This time frame puts a perspective on how long it takes for the human body to store excess glucose in their blood stream from a recent meal as glycogen for later use as fuel. [7] Biological RelevanceIn a healthy individual, insulin is secreted in response to high blood glucose levels is bound by insulin receptors throughout the body which initiates a robust physiological response. Without properly functioning insulin receptors that can respond to increases in insulin, and therefore glucose, medical intervention is necessary for survival. Type 1 diabetes is classified as "insulin dependent" and involves an inability for the body to produce insulin, resulting from damage or insufficiency in the Islets of Langerhans in the pancreas. Type 2 diabetes is classified as "insulin independent" and is the result of the body producing insufficient amounts of insulin, or not responding to the insulin. Type II DiabetesType II diabetes (T2D) is a chronic condition that affects 10 percent of the world's population [2]. T2D is characterized by insulin resistance and leads to high concentrations of glucose in the bloodstream. A type II diabetic produces insulin, but when the insulin molecule binds to the insulin receptor, the signal is not properly transmitted intracellularly [2]. Insulin resistance in routine type II diabetics is not associated with mutations of the insulin receptor gene, but instead, the signal being disrupted later in the pathway [2]. Mutations of the receptor gene are associated with more severe cases of insulin resistance, as seen in leprechaunism. Additionally, mutations of the insulin receptor can be fatal, as it is crucial for many cellular processes including gene expression, glucose homeostasis, and apoptosis [2]. The basis for insulin resistance in typical type II diabetics is complex and cannot yet be explained by one particular factor.[13] [2] There are a multitude of hypotheses which discuss the reasons for the establishment of type II diabetes.[2] [13] While the specifics of the development of T2D are beyond the scope of this page, the molecular causes for insulin resistance and T2D have been primarily attributed to the inhibition of key proteins involved in the insulin signaling and glucose transport pathway.[2] Alterations to the phosphorylation cascade of insulin signaling can be the result of changes within the cellular environment including lipotoxicity, inflammation, hyperglycemia, and the presence of reactive oxygen species (ROS).[2] On a macroscopic level, a variety of factors influence the cellular environment, and thus the risk for T2D. These factors include gestational environment, microbiome, genetics, diet, and energy expenditure.[13] Recent studies, which have evaluated the relationships between genetics and environmental factors in the progress of T2D, have shown that T2D is not uniform among the population and the biochemistry behind the development of risk factors varies for each patient.[13]
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References
Student Contributors
Abby Hillan
Alyssa Ritter
Andrew Scheel
Harrison Smith
Maxwell Todd
