The Binding of Insulin to the Human Insulin Receptor (IR) and its effects: A Brief Exploration
Introduction
Receptor tyrosine kinases are a large family which are involved with the regulation of many processes throughout an organism.1 The human insulin receptor (IR) is a transmembrane protein which belongs to this family of receptors and is coded with the INSR gene. IRs must be activated using ligands such as insulin, the hormone, and they also play a major role in the maintaining of glucose levels and the regulation of the metabolism of proteins, lipids and carbohydrates.2 The actions of these receptors are also found in studies regarding type 2 diabetes2, cancer3 and Alzheimer's disease4. There are many ligands which are able to bind to these receptors varying in their degrees of affinity, not only insulin but also insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2), these ligands act as agonists, with each one being found on a different chromosome.
Insulin receptor
The human IR consists of two different subunits, α and β, which are bound together with the help of disulphide bonds to form the heterotetrametric receptor, (αβ)2, made up of glycoproteins5. This receptor possesses a molecular weight of around 135,000. There are two different isoforms of the IR when it undergoes differential splicing to give, IR-A and IR-B. The way in which they differ is that their binding affinities for insulin-like growth factor, along with their differences in abundances in organs and tissues. However, they do share the same affinity for insulin.6 There are many methods in which the structure of the receptor was determined including the purification of the receptor and then characterisation of the protein and utilising antibodies and then analysing them using electrophoresis along with just examining the insulin-receptor complex using different characterisation methods.7 This heterotetrametric structure can be separated into two IR αβ monomers. In the extracellular portion of the receptor they each have two repeat domains which are leucine-rich (L1 and L2), one cysteine-rich region (CR) and three fibronectin type III domains (FnIII-1, FnIII-2 and FnIII-3).8 The fibronectin type III domains do not have disulphide bonding unlike type I and II.9 In between the α-β cleavage site, in the FnIII-2 domain there is an insert domain (ID) which consists of 120 residues. This domain shares the α-β proteolytic processing site alongside the intra-monomer disulphide bond which links the α-monomer and β-monomer. The residues involved in this linkage are C647 and C860.10 There are also disulphide linkages found between in the α-monomers at C524 and C683, which stabilise the entire structure of the receptor.8 Now moving to the intracellular section of the receptor, from the C-terminus end there is a tyrosine kinase (TK) domain which is isolated from other regions with the help of the juxtamembrane region and the other C-terminus end tail.11
Insulin
Insulin is a vital hormone present in the endocrine system, consisting of fifty-one amino acids, which ensures that the blood glucose levels in the blood are regulated along with carbohydrate metabolism. It is secreted by the β cells in the pancreatic islets of Langerhans. When insulin is stored, it is found in a hexamer which is coordinated by a histidine on each of the monomers to two zinc ions in the centre of the hexamer. This structure however dissociates, to allow itself to bind to the IR, into a single insulin monomer. These monomers have no zinc ion and consist of two chains which are connected using three disulphide bridges. With one being intramolecular in chain A and the other being intermolecular between the two chains. However, insulin is still not in the correct conformation in order to bind to the receptor and it must change its structure to become active. If this conformation does not take place then there will be a steric clash between subunits and residues. The conformational changes that occur are a 10° rotation around the GlyB20 residue and a 50° turn around PheB24, which is referred to as the B26 turn. The B26 turn is stabilised by a hydrogen bond between TyrB26 and PheB24 and another hydrogen bond between TyrB26 and GlyB8. These intermolecular bonds allow the stabilisation of insulin into the active conformation. For stabilisation of insulin to be successful the N-terminus of the Chain A undergo the packing of ValA3 and IleA2 along with the presence of the intramolecular Chain A disulphide bond.
Ligand Binding
As said before, the receptor must be activated to carry out further reactions within the cells. The binding model for the insulin receptor suggests that there are two binding sites, site 1 and site 2, found on two different α-subunits. Once a ligand, either insulin or any insulin-like growth factor, binds to the first low-affinity site then it continues and binds to the second site on the other α-subunit, forming a ligand-IR complex.12The differences that the ligands have with each other in terms of binding to the sites is that they have varying affinities for the binding sites. The differences in binding affinities have been proven by the study of the ligand binding properties which when plotted produce curvilinear Scatchard plots. This curvilinearity shows the presence of both the low and high affinity sites. 13 A process that also occurs during the ligand binding is negative cooperativity, which describes the process that the high-affinity site can only be occupied if the low-affinity site is occupied and the complex formed can move from one side of the dimer to the other, as no ligand is able to bind to both sites simultaneously.12 The binding of a ligand causes the kickstart of the tyrosine kinase catalytic activity leading to the phosphorylation of some tyrosine residues along with some structural changes being induced to allow the entry of glucose into the cell. These responses to the binding of the ligands promote the transfer of the signals which are involved in blood glucose homeostasis.14
Insulin Binding
Once insulin undergoes all its conformations, it uncovers the site which is ready to be bound to the IR in a stable form. The site formed involves from Chain A: GlyA1, IleA2, ValA3, GlmA5 and TyrA19 and from Chain B: ValB12, LeuB11, PheB24 and PheB25. This site interacts with the IR using van der Waals interactions which occur once the IR residues interlink with the hydrophobic pocket formed by the residues which have been exposed. The aromatic side chains of insulin have an important role in allowing insulin to bind to the IR, which are shown by the aromatic ring of PheB24 projecting into the hydrophobic pocket exposed to allow an interaction (van der Waals) with Phe714 in the IR as well as its other interactions with ValB12, LeuB15 and TyrB26.
Conformational Change
Once a ligand binds to the receptor it is has been proven that there is a conformational change from the structure of the receptor before the addition of the ligand. The main observational difference between the two structures is that in the bound structure has a much more enhanced network of intermolecular electrostatic interactions. The main one being on the interface of the L2/FnIII-2 with the FnIII-2 residues Asp496, Arg498 and Asp499, and the L2 residues Arg454 and Glu453 where they are now capable of forming electrostatic interactions such as van der Waals after the slight conformation due to the binding of the ligand.8
Diabetes Mellitis
When the monitoring of the blood glucose levels is not properly managed it can lead to a very common and widespread disease called diabetes mellitis. This can be split into two different types: Type I which arises when the patient is young and happens due to the body's autoimmune system rejecting the insulin produced by the pancreatic skills, or that the insulin is mutated and can no longer be used as it is inactive as it cannot conform itself into the active form needed, and Type II which happens as an adult is caused as a resistance has formed against the action of insulin on its receptor. The results of this involve phosphorylation of the receptor and the insulin substrate to change the way in which they carry out insulin signalling, the way in which this type is treated is by altering lifestyle, medication and diet. A common medication which is prescribed to assist with type II diabetes is Metformin. Its main action is to decrease the overproduction of glucose by the liver to lower blood glucose levels in the blood. This is turn decreases insulin resistance and improves the reactivity of the body towards insulin, allowing the insulin your body makes to be used more effectively.
Conclusion
This essay touches on the structure of both insulin and the human insulin receptor and how they bind with one another along with the effects the different levels of insulin in the blood can have on a person's health. Another main focus of this essay is the way in which ligands bind to the receptor as well as more specifically how insulin as a ligand binds to it and the subsequent outcomes of this process. When looking into the future regarding the secretion of insulin and how it binds to the human IR, the possibility of creating artificial β-cells that secrete insulin may be a solution as bioengineers are working on producing such a result.
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