Student Projects for UMass Chemistry 423 Spring 2012-1

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Insulin Receptor

by Ryan Deeney, Jeffrey Boerth, Kate Liedell, Rebecca Bishop, Student Projects for UMass Chemistry 423 Spring 2012

Introduction

Insulin receptors are expressed at the cell surface as disulfide-linked homodimers composed of alpha/beta monomers(pdb code 3loh).

Drag the structure with the mouse to rotate

The insulin receptor is a tyrosine kinase, that is a type of ligand-activated receptor kinase. The crystallized protein, shown here, is the of the insulin receptor, as it is difficult to crystallize the protein and determine the structure when the greasy transmembrane portion of the protein is included. Four FAB antibodies (shown in peach, yellow, light green, and light blue) are attached to the protein to aid in crystallization. The insulin receptor is to the membrane at the beta strand, which extends through the cell membrane. The receptor is attached to the cell membrane by the beta strand, which extends through the membrane and into the interior of the cell and mediates activity by the addition of phosphate to tyrosines on specific proteins in cell.

Insulin receptors are found in many diverse organisms organisms, from cnidarians and insects to humans. In humans, correctly functioning insulin receptors are essential for maintaining glucose levels in the blood. The insulin receptor also has role in growth and development (through insulin growth factor II); studies have shown that signalling through IGF2 plays a role in the mediation embryonic growth. [1]

In everyday function, insulin binding leads to increase in the high-affinity glucose transporter (Glut4) molecules on the outer membrane of the cell in muscle and adipose tissue. Glut4 mediates the transport of glucose into the cell, so an increase in Glut4 leads to increased glucose uptake. Insulin has two different receptor-binding surfaces on opposite sides of the molecule, that interact with two different . The first binding insulin surface interacts with a site on the L1 module as well as a 120-amino-acid peptide from the insert in FnIII-2. The second binding site consists of resides on the C-terminal portion of L2 and in the FnIII-1 and FnIII-2 modules [2]. Binding sites are shown highlighted in both monomers of the biologically functional dimer. [3]

Maintaining appropriate blood glucose levels is essential for appropriate life-sustaining metabolic function, and insulin receptor malfunction is associated with several severe diseases. Insulin insensitivity, or decreased insulin receptor signalling, leads to diabetes mellitus type 2. Type 2 diabetes is also known as non-insulin-dependent or adult onset diabetes, and is believed to be caused by a combination of obesity and genetic predisposition. In type 2 diabetes, cells are unable to uptake glucose due to decreased insulin receptor signaling, which leads to hyperglycemia (increased circulating glucose). Type 2 diabetes can be managed with dietary and lifestyle modifications to aid in proper metabolism.

Mutations in both copies of the insulin receptor gene causes Donohue syndrome, which is also known as leprechaunism. Donohue syndrome is an autosomal recessive disorder that results in a totally non-functional insulin receptor. The disorder results in distorted facial features, severe growth retardation, and often death within a year.[4] A less severe mutation of the same gene causes a much milder form of the disease in which there is some insulin resistance but normal growth and subcutaneous fat distribution.[5]

Overall Structure

The Different Domains of the Insulin Receptor Ectodomain

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The ectodomain of the insulin receptor is a of 2 identical monomers. Each v-shaped is composed of 6 domains, three on each side of the V, shown in different colors. The red (L1) is involved in substrate binding. Its main feature is a 6 parallel stranded beta sheet. The orange (CR) is composed mostly of loops and turns. The yellow domain is a second , (L2) which contains a five parallel stranded beta sheet and several surface alpha helices. [6]

The next three domains are Fibronectin Type III domains. Fibronectin domains, characterized by beta sandwiches, are named after the protein fibronectin, which contains 16 of these domains.[7] The green FnIII-1 , contains one antiparallel and one mixed beta sheet. The blue FnIII-2 contains an insert domain of 120 residues. The purple FnIII-3 contains just four beta strands. Each domain occurs twice in the .[8]

The insert domain of the FnIII-2 domain separates the alpha and beta chains of each monomer. The alpha chain contains the L1, CR, L2, FnIII-1 domains and part of the FnIII-2 domain. The beta chain contains the rest of the FnIII-2 domain and the FnIII-3 domain. The insert domain starts and ends with a cleavage site where the chain is cut. The alpha and beta chains are then linked by a single between cysteines C647 and C860, leaving the insert domain as a separate peptide which forms disulphide bonds with cysteines in the FnIII-1 domain. The alpha chain lies completely on the exterior of the cell, while the end of the beta chain extends through the cell membrane and is involved in signaling.[9] This section of the beta chain, after the FnIII-3 domain in the sequence, is not shown in the structure which reflects only the . There are few interactions between the two legs of the monomer- just two salt bridges near the connection between the L2 and FnIII-1 domains. However there are many interactions between the two monomers including salt bridges and disulphide bonds.[10]

This structure is significant relative to previous structures for the protein because of the relative position of the L1 domains in the two monomers of the biological unit. In previously proposed structures, the L2, CR, and L1 domains formed a straight leg of the V similar to that of the fibronectin leg. With this model, it was thought that both L1 domains could bind to a single insulin molecule. With this folded over structure of the L2-CR-L1 leg, it is clear that this is not the case, as the L1 domains of each monomer face away from each other.[11]

Binding Interactions

Depicted here is the monomer form of human insulin. The hydrophilic residues are shown in purple and the hydrophobic residues are shown in gray.

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The insulin receptor's (IR) main ligand is insulin. As insulin binds to the IR, the IR is phosphorylated. Phosphorylation of tyrosine residues in the IR leads to activation of the kinase activity of the receptor, and to phosphorylation of the insulin receptor substrate 1 (IRS-1). This is the start of a signalling cascade that eventually leads to an increase in the glucose transporter (Glut-4) which has a high affinity for glucose molecules. This occurs mainly in muscle and adipose (fat) tissues where glucose uptake is most needed, and in the liver, which stores glucose in the form of glycogen or metabolizes it to fatty acids. This increase in Glut-4 causes an increase in glucose uptake from blood. Simply stated, 3loh is activated by insulin (IRS-1) which signals for an increase in Glut-4. Glut-4 finds its way to the cell surface where it can perform its function and transport glucose into the cell.

The binding mechanism and site are not completely understood. What is known, however, is that insulin is able to bind at two different locations on each monomer of the insulin receptor. Since there are two monomers in the biologically functional ectodomain, there are in total four locations available for binding and interaction. These locations are explained in the Introduction section of this page. Current literature describes the locations for insulin binding as follows: the region between L1 and Fn2 as site 1, the region involving L2, Fn1, and Fn2 as site 2. Based on knowledge of the structure of the insulin receptor (it is a dimer with anti-mirrored symmetry), one can see that the site 1 of one monomer will be adjacent to site 2 on the other. In order to eliminate confusion, most literature refer to the binding sites across from site 1 and site 2 as site 2' and site 1', respectively. One of the most popular theories that is used to explain insulin binding describes that two molecules of insulin must bind to the IR in order for it to become active and for the kinase cascade to initiate. In this case, binding of two insulin molecules would occur at sites 1/2' and 2/1'. This is only a theory, however, and none of these theories have been completely confirmed [12].

Recent research has indicated that it may be possible that both the structure of the insulin receptor and the structure of insulin itself may change upon binding. It is also thought that insulin may possess multiple surfaces that are capable of binding to the functional ectodomain.

In order to better understand the binding of the insulin receptor, it would make sense to observe its main ligand, . This green scene shows both the hydrophobic and hydrophilic residues. The binding surface is mostly comprised of residues that are hydrophobic.

Another interesting point to mention is that insulin in its can also interact with the binding sites available on the insulin receptor. Hexamers of insulin are found in the pancreas and help store insulin. They consist of 3 insulin dimers that are held together by 2 Zn ions.Upon creating the hexamer form, a new binding surface for insulin is created that exhibits normal binding at site 1. Binding does not occur at site 2 however, and thus the hexamer form of insulin does not activate the insulin receptor as does regular form of insulin. Here is a that highlights the second binding surface. The second binding surface is highlighted on one of the three dimers and involves a small group of specific residues: SerA12, LeuA13, GluA17, HisB10, GluB13, and LeuB17 [13].

Additional Features

Shown above is the Insert Domain in green within the homodimer

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Interestingly, the molecular basis as to how insulin binds to the insulin receptor substrate (IRS) is not yet fully understood. However, there has been some light shed on the kinase cascade pathway that insulin induces.

Recently, studies have shown that poor diet and increased sugar intake have led to a halt in the kinase cascade pathway, leading to what is now termed as insulin resistance.

Insulin Resistance: Happens when the cells essentially don't open the door when insulin comes knocking. When this happens, the body puts out more insulin to stabilize blood glucose in the body. This allows for a vicious cycle where the cells become more and more desensitized as the concentration of insulin increases to tackle the constant influx of glucose. This occurs when the insulin receptor cannot activate the glucose transporter (Glut-4) vesicles through the kinase cascade in order for Glut-4 to bind to the cell membrane and bring in glucose. [14]

Mechanism: When insulin binds it phosphorylates the IRS, leading to a kinase cascade pathway that ineveitably activates Glut-4 to bind to the cell membrane. Naturally, once the blood glucose has reached a normal level, the kinases are then dephosphorylated, which in turn slowly lowers the amount of glucose channels on the membrane surface. This is the normal negative feedback loop that takes place within the cell. However, when sugar intake is too high for too long, the amount of stored glucose (glycogen) will reach levels where the cell will try to stop the kinase pathway at any point necessary. At this point, the IRS will not be able to perform signal transduction even with the binding of insulin, proving insulin to be ineffective. In response to the increase in blood glucose, the Pancreas cranks out insulin in an attempt to lower blood glucose, when the binding interaction with IRS will essentially do nothing. In the end, all of the kinases become stuck in the dephosphorylated state even with high concentrations of insulin, and no glucose can be stored. The traffic jam will either kill the cell, or if glucose intake recedes, the cell can try to restore itself to its normal feedback loop. [15]

So what's the cure?

Extensive research has been conducted to see if the IRS can bind to other proteins which can then induce the kinase cascade pathway. In one experiment, the within the IRS has shown to exhibit binding to the active sites of the IRS. It does so through what has been hypothesized to be between the two monomers in the homodimer. The actual peptide connecting one piece of the insert domain to the other has yet to be resolved, however the binding portion of the insert domain has been to be at Site 1, between the L1 and FnIII-2 domains. Since insulin binds to Site 1 as well, it is also hypothesized that the binding portion of the insert domain competitively binds with the insulin protein because of its mimical structure to insulin. This is quite an important discovery, because a compound that can mimic the structure of insulin could have a higher affinity for the IRS, which could activate the signal transduction pathway. Therefore, the IRS may provide a target for a drug, perhaps also achievable in part by molecules the size of antibiotics. [16]

Best solution? Eat Healthy! Reducing sugar intake by eating less sweets should cause a break down of excess glycogen, returning cells to normal over time.

Credits

Introduction - Rebecca Bishop

Overall Structure - Kathryn Liedell

Drug Binding Site - Ryan Deeney

Additional Features - Jeffrey Boerth

References

  1. ↑ Kitamura T, Kahn CR, Accili D. Insulin receptor knockout mice. Annu Rev Physiol. 2003;65:313-32. Epub 2002 May 1. PMID:12471165 doi:10.1146/annurev.physiol.65.092101.142540
  2. ↑ Fried R. A literary look at contemporary society. Ohio Med. 1989 May;85(5):393-5. PMID:2657531
  3. ↑ Whittaker L, Hao C, Fu W, Whittaker J. High-affinity insulin binding: insulin interacts with two receptor ligand binding sites. Biochemistry. 2008 Dec 2;47(48):12900-9. PMID:18991400 doi:10.1021/bi801693h
  4. ↑ Longo N, Wang Y, Smith SA, Langley SD, DiMeglio LA, Giannella-Neto D. Genotype-phenotype correlation in inherited severe insulin resistance. Hum Mol Genet. 2002 Jun 1;11(12):1465-75. PMID:12023989
  5. ↑ al-Gazali LI, Khalil M, Devadas K. A syndrome of insulin resistance resembling leprechaunism in five sibs of consanguineous parents. J Med Genet. 1993 Jun;30(6):470-5. PMID:8326490
  6. ↑ McKern NM, Lawrence MC, Streltsov VA, Lou MZ, Adams TE, Lovrecz GO, Elleman TC, Richards KM, Bentley JD, Pilling PA, Hoyne PA, Cartledge KA, Pham TM, Lewis JL, Sankovich SE, Stoichevska V, Da Silva E, Robinson CP, Frenkel MJ, Sparrow LG, Fernley RT, Epa VC, Ward CW. Structure of the insulin receptor ectodomain reveals a folded-over conformation. Nature. 2006 Sep 14;443(7108):218-21. Epub 2006 Sep 6. PMID:16957736 doi:10.1038/nature05106
  7. ↑ Kornblihtt AR, Umezawa K, Vibe-Pedersen K, Baralle FE. Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 1985 Jul;4(7):1755-9. PMID:2992939
  8. ↑ McKern NM, Lawrence MC, Streltsov VA, Lou MZ, Adams TE, Lovrecz GO, Elleman TC, Richards KM, Bentley JD, Pilling PA, Hoyne PA, Cartledge KA, Pham TM, Lewis JL, Sankovich SE, Stoichevska V, Da Silva E, Robinson CP, Frenkel MJ, Sparrow LG, Fernley RT, Epa VC, Ward CW. Structure of the insulin receptor ectodomain reveals a folded-over conformation. Nature. 2006 Sep 14;443(7108):218-21. Epub 2006 Sep 6. PMID:16957736 doi:10.1038/nature05106
  9. ↑ Smith BJ, Huang K, Kong G, Chan SJ, Nakagawa S, Menting JG, Hu SQ, Whittaker J, Steiner DF, Katsoyannis PG, Ward CW, Weiss MA, Lawrence MC. Structural resolution of a tandem hormone-binding element in the insulin receptor and its implications for design of peptide agonists. Proc Natl Acad Sci U S A. 2010 Apr 13;107(15):6771-6. Epub 2010 Mar 26. PMID:20348418
  10. ↑ McKern NM, Lawrence MC, Streltsov VA, Lou MZ, Adams TE, Lovrecz GO, Elleman TC, Richards KM, Bentley JD, Pilling PA, Hoyne PA, Cartledge KA, Pham TM, Lewis JL, Sankovich SE, Stoichevska V, Da Silva E, Robinson CP, Frenkel MJ, Sparrow LG, Fernley RT, Epa VC, Ward CW. Structure of the insulin receptor ectodomain reveals a folded-over conformation. Nature. 2006 Sep 14;443(7108):218-21. Epub 2006 Sep 6. PMID:16957736 doi:10.1038/nature05106
  11. ↑ McKern NM, Lawrence MC, Streltsov VA, Lou MZ, Adams TE, Lovrecz GO, Elleman TC, Richards KM, Bentley JD, Pilling PA, Hoyne PA, Cartledge KA, Pham TM, Lewis JL, Sankovich SE, Stoichevska V, Da Silva E, Robinson CP, Frenkel MJ, Sparrow LG, Fernley RT, Epa VC, Ward CW. Structure of the insulin receptor ectodomain reveals a folded-over conformation. Nature. 2006 Sep 14;443(7108):218-21. Epub 2006 Sep 6. PMID:16957736 doi:10.1038/nature05106
  12. ↑ Ward CW, Lawrence MC. Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor. Bioessays. 2009 Apr;31(4):422-34. PMID:19274663 doi:10.1002/bies.200800210
  13. ↑ Ward CW, Lawrence MC. Ligand-induced activation of the insulin receptor: a multi-step process involving structural changes in both the ligand and the receptor. Bioessays. 2009 Apr;31(4):422-34. PMID:19274663 doi:10.1002/bies.200800210
  14. ↑ Cusi K, Maezono K, Osman A, Pendergrass M, Patti ME, Pratipanawatr T, DeFronzo RA, Kahn CR, Mandarino LJ. Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle. J Clin Invest. 2000 Feb;105(3):311-20. PMID:10675357 doi:10.1172/JCI7535
  15. ↑ Zick, Y. Biochemical Society. 2004, 32, 812-816
  16. ↑ Smith BJ, Huang K, Kong G, Chan SJ, Nakagawa S, Menting JG, Hu SQ, Whittaker J, Steiner DF, Katsoyannis PG, Ward CW, Weiss MA, Lawrence MC. Structural resolution of a tandem hormone-binding element in the insulin receptor and its implications for design of peptide agonists. Proc Natl Acad Sci U S A. 2010 Apr 13;107(15):6771-6. Epub 2010 Mar 26. PMID:20348418

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