Insulin signal transduction pathway

Insulin and receptor (RTK class II)

• Insulin
• Insulin receptor

The insulin receptor (IR) is a dimer of made of 2 and 2 . Within the extracellular ectodomain, there are 4 potential that can interact with insulin on the extracellular side of the membrane.

The make up the extracellular domain of the IR and are the sites of insulin binding. The α-subunit is comprised of 2 Leucine rich domains (L1 & L2), a Cysteine rich domain (CR), and a . 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 α-subunits are held together by a disulfide bond between on each α-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 α-subunits, and . 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 β-sheet of the FnIII-1 domain and lack of surface area decreases the likelihood of their binding site becoming occupied as quickly.

The spans from the extracellular domain across the TM region and into the intracellular portion of the IR. The β-subunit is composed of part of fibronectin domain III-2 and all of Fibronectin domain III-3. The β-subunit's FnIII-3 domain has links through the TM region into the intracellular part of the membrane. Cryo-EM provided clear representations of the FnIII-2 and FnIII-3 domains, but are missing the TM and intracellular regions. Although the FnIII-3 domain is connected to the TM and intracellular regions, the active conformation likely extends all the way to the tyrosine kinase domain region (4xlv).

The α and β subunits of the extracellular domains fold over one another and form a when the IR is inactivated. Upon activation, the extracellular domain undergoes a conformational change and forms a . An additional component to the ectodomain is . Each of the dimers has an α-CT helix. The α-CT helix is a single α-helix that plays an important role in insulin binding and stabilization of the "T" shape activated conformation. α-CT interacts with a leucine-rich region of the α subunit and a fibronectin type III region of the β subunit to form the insulin binding sites known as .

The structure of the extracellular domain is stabilized through multiple disulfide bonds. The α-subunits are linked through 2 disulfide bonds, with the main one being between of 2 adjacent α-subunits. of both α-subunits are also held together with a disulfide bond. The α-subunit is also attached to the β-subunit by a disulfide bond between the . The IR unit has 4 separate sites for the insulin binding. There are 2 pairs of 2 identical binding sites referred to as and .

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 of the IR FnIII-1 domain. 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, to allow the hydrophobic part of the side chain to interact with the other hydrophobic residues. The α-subunits also have significant that help maintain a compact binging site. At sites 2 and 2', the major residues contributing to these hydrophobic interactions are the .

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). The binding interactions of the insulin molecules in sites 1 and 1' are facilitated by hydrophobic residues of an of the IR. The insulin molecules in sites 2 and 2' primarily interact with the residues that comprise some of the of the IR.

At , a occurs between 3 critical parts of the α subunits of the IR. 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 IR and their interaction involves the following residues: and the . This duo then interacts with the L1 region, specifically ARG14, creating an ideal for the insulin ligand. The FnIII-1 and α-CT are interacting from the 2 different α-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 2 subunits of the IR that maintains and stabilizes the T-shape activation state for the rest of the downstream signaling to occur.

It has been hypothesized that activation of the IR can change based on the concentration of insulin. These structures of the IR have demonstrated that at least 3 insulin molecules have to bind to the IR to induce the active conformation, as binding of 2 insulin molecules is insufficient to induce a full conformational change. However, this conclusion has not yet been widely confirmed. In low concentrations of insulin, the IR may not require binding of 3 insulin molecules in order to exhibit activation. Rather, the level of activity will change in accordance to the availability of insulin. When higher concentrations of insulin are present, the conformational difference between the 2-insulin-bound state and the 3-insulin-bound state is drastic as the IR transitions from the inactive to the active . However, in conditions of low insulin availability, the 2-insulin-bound state may be enough to induce partial activation of the receptor.

The conformational change between the inverted, inactive and the active of the IR is induced by insulin binding. The T shape conformation is well observed in the α subunit. It is horizontally composed of L1, CR (including the ), and L2 domains and vertically composed of the FnIII-1, 2, and 3 domains. The proper conformational change of the ectodomain of the IR is crucial for transmitting the signal into the cell. The movements extracellularly cause the 2 receptor tyrosine kinase domains intracellularly to become close enough to each other to autophosphorylate. This autophosphorylation activates the tyrosine kinase domain, initiating intracellular insulin signaling cascades.

When an insulin molecule binds to site 1 of the α-subunit, the respective protomer is recruited and a slight inward movement of the of the β-subunit is initiated. This is accomplished by the formation of several salt bridges, specifically between . Binding of insulin to both protomers establishes a full activation of the IR. 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. The conformational change and "hinge motion" between the inactive and active forms of an IR protomer. Upon insulin binding, the β subunits of the inactive form, shown in blue, are "swung" inward to the active form, shown in orange. When the receptor is in an , the FnIII-3 domains are separated by about 120Å. 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 conformation. As the fibronectin type III domains of the β subunit swing inward, the α 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 binding site. For the tripartite interface to form, the α subunits of each protomer must undergo a "folding" motion.

(3bu5).

TK domain of IR contains an activation loop and a catalytic loop and . The bound IR substrate 2 peptide tyrosine is the phosphorylated residue^[1].

In type 2 diabetes, the TK domain is thought to be down-regulated through phosphorylation of by protein kinase C^[2].

. Water molecules shown as red spheres.

• Diabetes & Hypoglycemia
  
• Insulin (Hebrew)
  
• Insulin glargine
  
• Insulin and pro-insulin (Hebrew)
  
• Insulin-Degrading Enzyme
  
• Molecular_Playground/Insulin
  
• Insulin Structure & Function