User:R. Jeremy Johnson/Insulin Receptor: Difference between revisions

The insulin receptor binds the insulin hormone and initiates a cascade of events within the cell. The receptor resides within the [http://en.wikipedia.org/wiki/Cell_membrane plasma membrane] of insulin targeted cells. These cells are found in various organs, such as the liver, and tissues, including skeletal muscle and adipose.<ref name="Boucher"> PMID: 24384568</ref> The insulin receptor is activated by multiple insulin molecules binding to various sites on the receptor.<ref name="Uchikawa"> DOI:10.7554/eLife.48630</ref> Once activated, the receptor serves as the gateway for the regulation of various cellular processes including glucose transport, glycogen storage, [http://en.wikipedia.org/wiki/Autophagy autophagy], [http://en.wikipedia.org/wiki/Apoptosis 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.<ref name="Scapin"> DOI:10.1038/nature26153</ref>  

Through recent [http://en.wikipedia.org/wiki/Transmission_electron_cryomicroscopy 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.

<scene name='83/839263/Insulin_molecule/3'>Insulin</scene> is a [http://en.wikipedia.org/wiki/Hormone hormone] made of two separate amino acid chains that are bound by multiple disulfide bonds. Insulin is synthesized and secreted from the [http://en.wikipedia.org/wiki/Pancreatic_islets 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.<ref name="Meyts"> DOI:10.1007/BF00400837</ref> <ref name="Uchikawa" /> <ref name="Schäffer"> PMID: 1472036</ref> Current hypotheses propose that the receptor is fully activated only after multiple insulin molecules are bound.<ref name="Uchikawa" /> 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 [http://en.wikipedia.org/wiki/Insulin_receptor_substrate Insulin Receptor Substrate] (IRS).<ref name= "White"> PMID: 8276779</ref> The transport of extracellular glucose into the cell allows this glucose to be converted to [http://en.wikipedia.org/wiki/Glycogen glycogen] for storage and later usage.

The <scene name='83/832953/Alpha_subunits/5'>alpha subunits</scene> make up the extracellular domain ([http://en.wikipedia.org/wiki/Ectodomain 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 <scene name='83/832953/Alpha_c_helix/6'>an α-chain C-terminal helix (α-CT)</scene> (Figure 1).<ref name="Scapin"> PMID 29512653 </ref> α-CT has a unique 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 [http://en.wikipedia.org/wiki/Disulfide disulfide bond] between <scene name='83/832953/Cysteine_bond/2'>cysteine residues</scene> 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, <scene name='83/832953/Sites_1_and_1_prime_location/17'>sites 1 and 1'</scene> and <scene name='83/832953/Sites_2_and_2_prime_location/13'>sites 2 and 2'</scene> (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 for insulin binding than 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.<ref name="Uchikawa"> DOI 10.7554/eLife.48630 </ref>

The <scene name='83/832953/Beta_subunits/4'>beta subunits</scene> 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 [http://en.wikipedia.org/wiki/Fibronectin fibronectin] domain III-2 and all of Fibronectin domain III-3.<ref name="Scapin" /> 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 <scene name='83/839263/T-shape/4'>T-shape</scene> conformation (Figure 3) likely extends all the way to the tyrosine kinase domain region (see [http://www.rcsb.org/structure/4XLV PDB 4XLV]).<ref name= "Cabail"> DOI: 10.1038/ncomms7406 </ref>  

The alpha and beta subunits of the extracellular domains fold over one another and form a <scene name='83/839263/V_shape/3'>"V" shape</scene> when the insulin receptor is inactivated. Upon activation, the extracellular domain undergoes a conformational change and forms a <scene name='83/839263/T-shape/4'>"T" shape</scene>. An additional component to the [http://en.wikipedia.org/wiki/Ectodomain ectodomain] is <scene name='83/839263/Alpha-ct/2'> α-CT</scene>.<ref name= "Uchikawa" /> Each of the dimers has an α-CT. The α-CT is a single alpha-helix and it plays an important role in insulin binding and stabilization of the "T" shape activated conformation. The α-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 <scene name='83/839263/Insulin_molecules_at_site_1/1'>site 1 and site 1'</scene>.<ref name="Uchikawa" />

The insulin molecules bind to these sites mostly through [http://en.wikipedia.org/wiki/Hydrophobic_effect hydrophobic interactions], with some of the most crucial residues at sites 1 and 1' being between <scene name='83/839263/Residues_of_site_1_binding/8'>Cys A7, Cys B7, and His B5 of insulin and Pro495, Phe497, and Arg498</scene> of the insulin receptor FnIII-1 domain.<ref name="Uchikawa" /> Despite some of the residues included being charged, the main interactions are still hydrophobic in this binding site. For example, due to arginine carrying its positive charge at the end of the side chain, <scene name='83/839263/Arginine_bending/1'> the side chain is bent</scene> to allow the hydrophobic part of the side chain to interact with the other hydrophobic residues. The alpha subunits also have significant <scene name='83/832953/Cysteine_bond/3'>disulfide linkages</scene> that help maintain a compact binging site. At sites 2 and 2', the major residues contributing to these hydrophobic interactions are the <scene name='83/839263/Site_2_residues_hydrophobic/4'>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</scene>.<ref name="Uchikawa" />  

 

Sites 1 and 1' have a higher binding affinity than sites 2 and 2' due to site 1 having a larger surface area (706 Ų) exposed for insulin to bind to compared to site 2 (394 Ų).<ref name="Uchikawa" /> The binding interactions of the insulin molecules in sites 1 and 1' are facilitated by hydrophobic residues of an <scene name='83/839263/Insulin_bound_to_site_1/4'>alpha-helix</scene> of the insulin receptor. The insulin molecules in sites 2 and 2' primarily interact with the residues that comprise some of the <scene name='83/839263/Insulin_in_site_2_with_beta_sh/7'>beta-sheets</scene> of the insulin receptor.  

At <scene name='83/832953/Sites_1_and_1_prime_location/17'>binding sites 1 and 1'</scene>, a <scene name='83/832953/Tripartite_interaction/8'>tripartite interaction</scene> occurs between three critical parts of the alpha subunits of the insulin receptor.<ref name="Uchikawa" /> 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 come into close proximity during the conformational change of the insulin receptor and their interaction involves the following residues: <scene name='83/832953/Alpha_ct_and_fniii-1/7'>ASP496, ARG498, and ASP499 on the FnIII-1 domain</scene> and the <scene name='83/832953/Alpha_ct_and_fniii-1/9'>LYS703, GLU706, and ASP707 on the α-CT domain</scene>. This duo then interacts with the L1 region, specifically ARG14, creating an ideal <scene name='83/832953/Tripartite_interaction/9'>binding site</scene> 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 α-CT, the 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.<ref name="Uchikawa" />  

It has been hypothesized that activation of the insulin receptor can change based on the concentration of insulin. These recent cryo-EM structures of the insulin receptor have demonstrated that at least three insulin molecules have to bind to the insulin receptor to induce the active <scene name='83/839263/T-shape/4'>"T" shape</scene> conformation, as binding of two insulin molecules is insufficient to induce a full conformational change.<ref name="Uchikawa" /> However, this conclusion has not yet been widely confirmed.<ref name="Uchikawa" /> 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.<ref name="Uchikawa" /> 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 <scene name='83/839263/V_shape/3'>"V" shape</scene> to the active <scene name='83/839263/T-shape/4'>"T" shape</scene>.<ref name="Uchikawa" /> However, in conditions of low insulin availability, the two-insulin-bound state may be enough to induce partial activation of the receptor.<ref name="Uchikawa" />

The conformational change between the inverted, inactive <scene name='83/839263/V_shape/3'>"V" shape</scene> and the active <scene name='83/839263/T-shape/4'>"T" shape</scene> 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 <scene name='83/832953/Alpha_c_helix/9'>α-CT chain</scene>), 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 [http://en.wikipedia.org/wiki/Autophosphorylation autophosphorylate].<ref name="Boucher" /> This autophosphorylation activates the tyrosine kinase domain, initiating intracellular insulin signaling cascades.<ref name="Boucher" />

When an insulin molecule binds to site 1 of the alpha subunit, the respective protomer is recruited and a slight inward movement of the <scene name='83/839263/Fniii_domains/1'>Fibronectin type III domains</scene> of the beta subunit is initiated. This is accomplished by the formation of several [http://en.wikipedia.org/wiki/Salt_bridge_(protein_and_supramolecular) salt bridges], specifically between <scene name='83/839263/Salt_bridges/1'>Arg498 and Asp499 of the FnIII-1 and Lys703, Glu706, and Asp707 of the alpha-CT</scene>.<ref name="Uchikawa" /> 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.<ref name="Uchikawa" /> 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 <scene name='83/832953/Inactive_insulin_receptor/6'>inverted V shape</scene>, the FnIII-3 domains are separated by about 120Å.<ref name= "Mckern"> PMID: 16957736</ref> 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, this conformation change brings the FnIII-3 domains within 40Å of each other to induce the <scene name='83/832953/Ir_dimer_t_state/4'>T shape</scene> conformation.<ref name="Uchikawa" /> <ref> DOI 10.1038/s41467-018-06826-6</ref> 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 α-CT and the FNIII-1 domains of the other protomer to form the <scene name='83/839263/Tripartite_interface/2'>tripartite interface</scene> binding site.<ref name="Uchikawa" /> For the tripartite interface to form, the alpha subunits of each protomer must undergo a "folding" motion. While snapshots of various conformational states of the insulin receptor have been captured, the complex dynamics of the insulin receptor conformational changes upon insulin binding are still being actively investigated.<ref name="Uchikawa" />

In a healthy individual, insulin secretion and binding to the insulin receptor initiates a robust physiological response. Improper insulin signaling leads to multiple disease states, including diabetes. [http://en.wikipedia.org/wiki/Type_1_diabetes 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. [http://en.wikipedia.org/wiki/Type_2_diabetes 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.   

[http://en.wikipedia.org/wiki/Type_2_diabetes Type II diabetes] (T2D) is a chronic condition that affects 10 percent of the world's population.<ref name="Boucher" /> 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. 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. Mutations of the receptor gene are associated with more severe cases of insulin resistance, as seen in [http://en.wikipedia.org/wiki/Donohue_syndrome 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. The basis for insulin resistance in typical type II diabetics is complex and cannot yet be explained by one particular factor.<ref name="Boucher" /> <ref name="Franks"> DOI:10.1126/science.aaf5094</ref>