Insulin Receptor Substrate Proteins

 
 
Structure and Overview
Function and Role
References

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Structure and Overview

 
OVERVIEW


  • Diabetes Mellitus is a chronic condition characterized as hyperglycemia (high levels of sugar in blood).  Continuing increments of blood glucose levels increase the risk of diabetes-related complications such as kidney damage, vision loss, heart disease, and foot ulcers.  Glucose is an essential fuel for the cells of the body, but only a normal amount of glucose in the blood circulation is necessary for the proper functioning of body organs.  The major sources of sugar come from carbohydrates in food and the storage form of glucose (glycogen) in the liver.  Before glucose can be utilized it must be transported from the bloodstream to the inside of the cells.  Such transport occurs through the use of insulin, a natural hormone produced by the pancreas.  The main job of insulin is to regulate the glucose transport into the cells by lowering the level of blood glucose.  [2] 
  • There are two major types of diabetes:  type 1 diabetes and type 2 diabetes.  With type 1 diabetes, heprglycemia develops because the pancreas cannot produce insulin.  This type of diabetes usually appears in childhood or young adulthood.  In type 2 diabetes, the pancreas is capable of producing insulin, but it cannot adequately meet the body's demands.  The problem is that the body does not respond to the insulin appropriately, which in turn leads to less glucose being absorbed by the cells and results in abnormally elevated blood glucose levels.  After overworking the pancreas for a number of years, the pancreas may eventually fail and exhaust its ability to produce insulin, which at this point a person with type 2 diabetes may require insulin therapy. [2]
  • Insulin also has dramatic effects on human embryonic development: matenal hyperinsulinaemia causes excess fetal growth, and insulin-resistant fetuses display growth retardation.  [3]
STRUCTURE
The actions of insulin are controlled through the activation of a heterotetrameric receptor which is found in the plasma membrane. The receptor gene is located on the short arm of human chromosome 19, is more than 150 kilobases in length and contains 22 exons.  The insulin receptor is a glycoprotein composed of two extracellular alpha-subunits and two transmembrane beta-subunits linked by disulfide bonds. The alpha-subunits contain the insulin-binding domain, and the intracellular portion of the beta-subunit contains the insulin-regulated tyrosine protein kinase (enzyme that catalyzes the transfer of a high-energy group from a donor--usually ATP--to an acceptor). 

Figure 1
Schematic diagram of the insulin receptor tetramer.  Membrane represented by a horizontal line.  [3]

Figure 2
Representation of the structure of the catalytic domain of the insulin receptor. [3]
 

When an insulin molecule is released by the beta cells of the pancreas and arrives at a cell, it binds onto the insulin receptor on the surface of most cells.  Once insulin has binded, the intrinsic phosphotransferase function of the insulin receptor (IR) beta-subunit is activated, resulting in the tyrosine phosphorylation of a number of intracellular proteins, including insulin receptor substrate (IRS)-1 through 4.  Insulin receptor substrate-1 is the most distinguished substrate whose phosphorylation of multiple tyrosine residues within the C terminus leads to the recruitment and subsequent activation of a number of scr-homology 2 (SH2) domain-containing proteins. [1] 

Figure 3 Human Insulin Hormone [8]    Figure 4 Insulin Receptor Tyrosine Kinase domain [6] 
 

Although the insulin receptor activates four substrate proteins, only IRS-1 and IRS-2 have been studied in depth.  IRS-1 and IRS-2 contain an amino-terminal pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain, and a carboxyl-terminal with multiple tyrosine phosphorylation motifs. [5] 

Figure 5  Ribbons and cylinder representation of the 1-IRS protein.  [4] 
 

The IRS proteins must co-localize with IR at or near the plasma membrane.  But phosphorylated IRS proteins may be able to relocate to alternative sites in the cell, whereas the movements of membrane-spanning receptors are restricted to plasma membrane and endosome compartments.  The four known IRS proteins have closely related targeting regions, which suggests common mechanisms for subcellular localization.  Each of targeting region is subdivided into a pleckstrin homology (PH) domain and a phosphotyrosine binding (PTB) domain.  The unique tandem arrangement, amino-terminal location, and high sequence homology of the PH and PTB domains defines the IRS protein family.  Sequence homology outside of the targeting region, in what is referred to as the activation domain, is much lower and limited to the short tyrosine-based motif that bind and activate Src homolgy 2 domain proteins. [10] 

Figure 6 IRS-1  PH-Ptb (N-terminal domain) [7]


Both the PH and PTB domain adopt the conserved PH domain fold, a seven-stranded, antiparallel beta-sandwich that is capped at one end by an alpha-helix.  No electron density is observed for the interdomain linker region which are residues 117-159.  The linker composition:  26 of the 43 residues are glycine, alanine, and serine.  The sequences for the PH and PTB domains are well conserved, unlike the interdomain linkers of the four IRS proteins which vary in legth, from 28 to 51 residues, and composition.  This suggests that this region of each IRS protein serves similarly as a flexible chain between PH and PTB domains.[10] 

Figure 7 


Sequence alignment of the amino-terminal domains of human IRS-1 and IRS-2 and rat IRS-3 and IRS-4.  Secondary structural elements of IRS-1 are shown above the alignments, and colored green (beta-sheets) or turquoise (alpha-helices).  Residues of the PTB domain that bind IR are labeled with red (phosphate binding) or black (all others) squares.  PH domain residues forming the cationic patch at its base are labeled with black squares. [10] 

 
 
 
 

The PTB domains of IRS-1, IRS-2, IRS-3, and IRS-4 are highly homologous (35% identity; 59-67% similarity) and are expected to have essentially identical structures.  The domains are closely associated with a contact surface buried between them that appears to be stabilized by ionic, hydrophobic, and hydrogen bonding interactions.  The PTB domain peptide binding site is fully exposed on the molecular surface, as is a large cationic patch at the base of the PH domain that is a likely binding site for the head groups of phosphatidylinositol phosphates.  Binding examinations confirm that phosphatidylinositol phosphates bind the PH domain, but not the PTB domain.  The structural data illustrates how the two binding domains might act cooperatively to effectively increase local insulin receptor substrate 1 concentration at the membrane and transiently fix the receptor and substrate, to allow multiple phosphorylation reactions to occur during each union. [10] 

Figure 8 

Structure of individual domains.  Ribbons (upper) and surface potential (lower) diagrams of the IRS-1 PTB and PH domains and the PLC PH domain are similarly oriented.  The IRS-1 PTB domain is bound to the IR juxtamembrane NPXpY peptide; the PLC PH domain is bound to Ins(1,4,5)P3.  An analogous pocket is at the base of the IRS-1 PH domain; the PTB domain has a distinct mode of binding.[10] 

The PH domain portion of the IRS-1 structure contains a short alpha1-helix, in addition to the seven beta-strands and the longer alpha2-helix common to PH domains.  The PH domain beta3/beta4 loop and the alpha1-helix within it pack against strands beta2, beta3, and beta4 of the bakc PH domain beta-sheet.  The PH domains of the other IRS proteins have very similar structues, sharing 23.5% identity and 40-50% similarity (see figure 7).[10] 

The PH and PTB domains are closely associated and arranged with the front sheet of the PH domain (beta5, beta6, and beta7) packed against the bakc of the PTB domain (beta1, beta2, beta3, and alpha2).  A hydrophobic patch between the domains is formed by the Phe-70 and Tyr-87 of the PH domain and Phe-160, Val-163, Arg-184, and Cys-186 of the PTB domain.  The interaction appears to be further stabilized by numerous potential hydrogen bonds, and potential salt brides between the side chains of Arg-75 and Glu-162; Lys-79 and both Asp-242 and Asp-241; and Arg-89 and Glu-200.  Conserved residues at the interface include Tyr-87m Oge-160, Val-163, Arg-184, and Cys-186 that form the hydrophobic patch between domains.[10] 

Figure 9 
 

 
Structure of the IRS-1 targeting domain. (A) Ribbon diagram of the PH-PTB structure, with beta-sheets shaded green, alpha-helices in turquoise, 310 turns colored indigo, and intervening coils or loops in brown.  (B) The PH/PTB domain interface viewed as an open book.  The PH and PTB domains each rotated 90 degrees, relative to their orientations in A, but in opposite directions to expose the buried surface between them.  Elements of secondary structure and contact residues are labeled.[10] 
 
 
 
 

 

 
 
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Function and Role
References

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Function and Role
To understand how the Insulin Receptor Substrate Proteins play such an important role in biometabolism, an understanding of the function of Insulin is first required: 

Functions of Insulin

  • Insulin promotes the storage of glucose in the form of glycogen.
  • Insulin promotes the biosynthesis of nucleic acids and protein.
  • Insulin increases uptake of glucose in the muscles and adipose tissue.
  • Insulin activates glycolysis in the liver.
Actions of Insulin
  • Insulin molecules exert their effect by binding to, and activating the insulin receptors.  These insulin receptors are part of a class of cell surface receptors that exhibit intrinsic tyrosine kinase activity.
  • Once the insulin receptor has been activated, phosphorylation events occur that lead to an increase in glucose storage and consequently a decrease in blood glucose levels.
  • For more information on insulin and its effects, the following web site may be helpful: THCME Medical Biochemistry Page
Figure 1.
Figure 1.
This figure is a schematic representation of the signal transduction pathway following the binding and activation of insulin to the insulin receptor. [8]

Insulin Receptor Substrate Proteins (IRS)

The Insulin Receptor Substrate (IRS) Protein family is part of the insulin signaling pathway.  Therefore, these IRS proteins help mediate the metabolic actions of insulin.
The IRS proteins serve as substrates for both insulin and insulin-like growth factor-1 (IGF-1) receptors.

IRS 1,2,3, and 4 have similar overall structure - each contains an N-terminal PH domain (Figure 3) followed by an FFB domain, and a C-terminal with many tyrosine phosphorylation sites. [7]
IRS-2 is the most abundant IRS protein in beta cells, whereas IRS-3 is only found in adipocytes, and IRS-4 is expressed mainly in the pituitary and thyroid gland. [11]
IRS-1 is the most abundant IRS protein and it is present in all cells.

How IRS Proteins Interact With the Insulin Receptor

  • Both IRS-1 and IRS-2 contain an N-terminal pleckstrin homology (PH) domain, a phosphotyrosine binding (PTB) domain (Figure 2.) , and a C-terminal with multiple tyrosine phosphorylation motifs. [7]
  • The insulin receptor is a heterotetrameric transmembrane glycoprotein.  It contains two extracellular alpha-subunits which are linked by disulfide bridges to two transmembrane beta-subunits.
  • The two alpha-subunits contain the insulin binding site while the two beta-subunits contain the tyrosine kinase that undergoes autophosphorylation once the insulin receptor has been activated by insulin.
  • Within the intracellular portion of the beta-subunits, there are three important structural regions: a juxtamembrane (JM) region, a kinase region, and a carboxyl-terminal (CT) region. [13]
a)  IRS-1
  • IRS-1 interacts with the insulin receptor via its PTB domain.
  • The PH region of IRS-1 brings the insulin receptor and IRS-1 into close proximity, while the PTB domain interacts with the JM region of the insulin receptor.
b)  IRS-2
  • Recently, a third region was discovered in IRS-2 which contributes to the IRS and insulin receptor interaction.  The third region interacts with the tyrosine residues in the regulatory loop of the kinase domain of the insulin receptor.
  • Because this region was not found in IRS-1, the finding suggests that IRS-1 and IRS-2 play different roles in the insulin signaling pathway.
  • Therefore, IRS-2 binds to the tyrosine-phosphorylated insulin receptor through its PTB domain which binds to the insulin receptor beta-subunit, and through the new domain that was discovered consisting of amino acids 591 to 786. 
  • The new domain interacts with the catalytic domain of the insulin receptor, and this interaction appears to be a stronger interaction than the PTB domain interaction. 
  • Recent evidence suggests that the IRS-2 domain containing the amino acids 591-786 is the primary interaction domain and the PTB domain serves to stabilize the interaction of the IRS-2 and the insulin receptor. [5]
IRS Proteins also Interact with Insulin-Like Growth Factor-1 (IGF-1) Receptors and Interleukins

Figure 2.  These figure is an IRS-1 PTB domain complexed with a IL(interleukin)-4 receptor [15]

Figure 3.  These figures represent different models of the A chain, PTB Domain , of the IRS-1 protein. [9a]  The left figure is the ribbon model and the right figure is the cylinder model.

Function of IRS Proteins

  • After insulin has bound to the extracellular alpha-subunit of the insulin receptor,  the receptor undergoes autophosphorylation on several of the tyrosine residues.  Tyrosine then phosporylates several substrates including the insulin receptor substrate 1 (IRS-1).  There are at least three substrates of the insulin receptor kinase (IRS-1, IRS-2, and Shc) which can serve as intermediates between the receptor and downstream insulin-mediated events. [6]
  • The majority of evidence suggests that IRS-1 plays the dominant role in the insulin signaling pathway.
  • There are three tyrosine residues in the tyrosine kinase domain of the insulin receptor, and phosphorylation of all three is necessary to activate the tyrosine kinase activity and initiate correct signaling to effectors. [6]

    •  
    • Human IRS has 6 tyrosine residues in Tyr-Met-X-Met or Tyr-X-X-Met motifs.  Tyrosine phosphorylation of IRS-1 results in IRS-1 being bound by several Src homology 2 (SH2) domain-containing proteins (Figure 4.) which include phosphatidylinositol (PI) 3-kinase, as well as two SH2 linker proteins - Grb-2 and Nck(3,11). [3]
    • Present studies indicate that tyrosine phosphorylation of IRS-1 is important in initiating several biological responses such as stimulation of growth responses and stimulation of  glucose uptake into the cells.
    • The IRS proteins serves as the interface between the activated insulin and IGF receptor, and several different signaling proteins within the cell. [6]
    • IRS-1 associates with the regulatory subunits of PI-3-kinase, Grb-2, and Nck, following insulin receptor activation.
    • The use of IRS proteins instead of just the autophosphorylation sites to activate the signaling molecules provides for amplification of the signal.
    • In addition to this, the binding of tyrosine-phosphorylated IRS-1 to PI 3-kinase causes a three to five fold increase in the PI 3-kinses's enzymatic activity. [2]
    • The activated PI 3-kinase initiates several of the final effects of insulin.  Some of these effects include glucose uptake by the cell and activation of several different serine or threonine kinases. [2]
    The Importance of IRS Proteins
    • Experiments with mutant insulin receptors confirmed that IRS-1 phosphorylation plays an essential role in the mediation of the insulin signaling pathway. [6]
    • Insulin receptors with impaired autophosphorylation consequently fail to phosphorylate IRS-1, and insulin signaling to downstream metabolic and mitogenic cellular events has been compromised. [6]
    • In various experiments with mice, the IRS-1 protein was removed by targeted gene mutation.  These mice showed a 50% reduction in intrauterine growth, impaired glucose tolerance, as well as a decrease in insulin-stimulated glucose uptake. [14]
    • In these experiments, there was evidence that IRS-2 compensated for the loss of IRS-1, however, this compensation was limited because there is an IRS-1 dependent and IRS-1 independent pathway of insulin signaling.
    • IRS-1 is involved in regulation of insulin content and it is thought to be required for maintaining glucose stimulated insulin secretion. [4]
    • Because IRS-2 is the most adundant IRS protein in beta-cells, IRS-2 is essential for the compensatory Beta-cell hyperplasia to insulin resistance. [4]
    The Link Between IRS Proteins and Type-2 Diabetes
     
    • Type-2 diabetes is characterized by a resistance to insulin.  Insulin is still produced by the beta-cells of the pancreas, however there is a failure in the signal conduction pathway of insulin, and the final metabolic effects mediated by insulin are not achieved in the cell.
    • It has been proposed that the IRS proteins play a role in type-2 diabetes.
    • IRS-1 not only undergoes tyrosine phosphorylation, but it also undergoes serine/threonine phosphorylation.  When a cell is stimulated by insulin, an increase in serine/threonine phosphorylation of IRS-1 occurs.
    • Recent evidence has shown that excessive serine/threonine phosphorylation of IRS-1 is one mechanism that allows tumor necrosis factor to induce a resistance to insulin.
    • The excessive serine/threonine phosphorylation of IRS-1 has been achieved experimentally by exposing cells to high concentrations of insulin for long periods of time. [2]
    • When IRS-1 undergoes excessive serine/threonine phosphorylation, there is a decrease in the ability of the insulin receptor to induce tyrosine phosphorylation of IRS-1.  Consequently, this leads to a reduction in the ability of IRS-1 to interact with PI 3-kinase. [2]
    • The interaction of IRS-1 with PI 3-kinase is important because an activated PI 3-kinase is required to initiate several of the final effects of insulin.  One of the crusial effects is the stimulation of glucose uptake by the cell.
    • Experiments were performed on mice without IRS-1 proteins.  These mice were smaller than the wild-type mice and they did display mild hyperinsulinemia, however, none of the mice developed type-2 diabetes.  The mice did display reduced glucose uptake ability in the skeletal muscles and reduced glycogen synthesis, but because they did not develop type-2 diabetes, an IRS-1 deficiency is not sufficient for type-2 diabetes. [10]


    Figure 4. These figures represent different models of both the A and B chain,  pH-PTB (N-terminal) Domain, of the IRS-1 protein. [9b]  The left figure is the ribbon model and the right figure is the cylinder model.


    Figure 5.
    Figure 5. shows the structure of the tyrosine kinase c-Src highlighting the important domains and residues critical for regulation of Src and its family members. [12]
     
     
     
     

     

     
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    Structure and overview
    References

     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     
     

    References for Structure and Overview

     
    1.  Bernier, Michel, Hemanta K. Kole, Chahrzad Montrose-Rafizadeh, and Sutapa Kole.  "Discrete Region of the Insulin Receptor Carboxyl Terminus Plays Key Role in Insulin Action."  Journal of Cellular Biochemistry April 2000 vol. 78 pp. 160-169.

    2.  http://www.medicinenet.com/Script/Main/art.asp MNI&articlekey 11013

    3.  White, M.F. "The Insulin Signalling System and the IRS Proteins."  Diabetologia (1997) 40:S2-S17.

    4.  http://www.rcsb.org/pdb/cgi/explore.cgi?pid=21905973740997&pdbId=1IRS 

    5.  Clark, Sharon F., Juan Carlos Molero, and David E. James.  "Release of Insulin Receptor Substrate Proteins from an Intracellular Complex Coincides with the Development of Insulin Resistance.  Journal of Biological Chemistry 11 Feb 2000 vol. 275 issue 6 3819-3826.  JBC Online http://www.jbc.org/cgi/content/full/275/6/3819

    6.  http://www.rcsb.org/pdb/cgi/explore.cgi?pid=27660973743906&page=0&pdbId=1IR3

    7.  http://www.rcsb.org/pdb/cgi/explore.cgi?pid=27660973743906&page=0&pdbId=1QQG

    8.  http://www.rcsb.org/pdb/cgi/explore.cgi?pid=4972973748200&page=0&pdbId=1BEN

    9. Travis, J.  "Diabetic Mice Cast Suspicion on Protein."  Science News 28 Feb 1998 v153 n9 p135.

    10. Dhe-Paganon, Sirano, Elizabeth A. Ottinger, Robert T. Nolte, Michael J. Eck, and Steven E. Shoelson.  "Crystal Structure of the Pleckstrin Homology-Phosphotyrosine binding (PH-PTB) Targeting Region of Insulin Receptor Substrate 1."  Journal of Biochememistry.  20 July 1999 Vol 96 Issue 15 pp. 8378-8383.

     
     
     
     
    References for Function and Role

     
    1.  Danielsen, Anne G., Feng Liu, Yoichi Hosomi, Kozui Shii, and Richard A. Roth.  Activation of 
    proteins Kinase C alpha Inhibits Signaling by Members of the Insulin Receptor Family.  Journal of Biological Chemistry.  September 2000,  vl 270,  pp 21600-21605.

    2.  De Fea, Kathryn, and Richard A. Roth. Protein Kinase C Modulation of Insulin Receptor Substrate-1 Tyrosine Phosphylation Requires Serine 612.  Biochemistry.  May 1997,  vl 42, 
    pp 12939-12947. 

    3.  Esposito, Diana, Yunhua Li, Lina Cong, Alessandro Cama, and Michael J. Quon.  [Tyr.sup.612] and [Tyr.sup.632] in IRS-1 Are Sufficient for Full Activation of Insulin-Stimulated PI-3-Kinase Activity and Translocation of GLUT4 in Rat Adipose Cells. Diabetes.  May 2000,  vl 49,  p A237.

    4.  Eto, Kazuhiro, et.al.  Role of IRS1/2 and PI-3-Kinase Pathway in the Regulation of [Beta]-Cell Mass and Glucose-Stimulated Insulin Secretion.  Diabetes.  May 2000,  vl 49,  p A45.

    5.  Sawka-Verhelle, Dominique, Sophie Tartare-Deckert, Morris F. White, and Emmanuel Va.  Insulin Receptor Substrate-2 Binds to the Insulin Receptor Through Its Phosphotyrosine-binding Domain and Through a Newly Identified Domain Comprising Amino Acids 591_786.  Journal of Biochemistry.  March 1996,  vl 271,  pp 5980-5983.

    6.  Krook, Anna, David E. Moller, Karim Dib, and Stephan O'Rahilly. Two Naturally Occuring Mutant Insulin Receptors Phosphorylate Insulin Receptor Substrate-1 (IRS-1) But Fail to Mediate the Biological Effects of Insulin.  Journal of Biological Chemistry.  March 1996,  vl 271, 
    pp 7134-7140.

    7.  Tsuruzoe, Kaku, Renee Emkey, Kristina M. Kriauciunas, and C. Ronald Kahn.  Impact of IRS-3 or IRS-4 Expression on IGF-1 Signaling Pathway in Embryonic Fibroblast Cells.  Diabetes.  May 2000,  vl 49,  pA336.

    8.  http://web.indstate.edu/thcme/mwking/diabetes.html
    Michael W. King, Ph.D / Medical Biochemistry / Terre Haute Center for Medical Education.

    9a.  http://www.rcsb.org/pdb/
    Zhou, M. M., Huang, B., Olejniczak, E. T., Meadows, R. P., Shuker, S. B., Miyazaki, M., Trub, T., Shoelson, S. E., Fesik, S. W.: Structural basis for IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain. Nat Struct Biol 3 pp. 388 (1996)  [ Medline ]

    9b.  http://www.rcsb.org/pdb/
    Dhe-Paganon, S., Ottinger, E. A., Nolte, R. T., Eck, M. J., Shoelson, S. E.: Crystal Structure of the Pleckstrin Homology-Phosphotyrosine Binding (Ph-Ptb) Targeting Region of Insulin Receptor Substrate 1 Proc. Nat .Acad. Sci. USA 96,   pp 8378 (1999)  [ Medline ]

    10.  Smith-Hall,  Jennifer, Sebastian Pons, Mary Elizabeth Patti, Deborah J. Burks, Lynne Yenush, Xiao Jian Sun, C. Ronald Kahn, and Morris F. White.  The 60 kDa Insulin Receptor Substrate Functions Like and IRS Protein in Adipose Cells.  Biochemistry.  December 1996, vl 27, 
    pp 8304-8310. 

    11.  Bektas,  Arsun, James H. Warram, Morris F. White, Andrzej S. Krolewski, and Alessandro Doria.  Exclusion of Insulin Receptor Substrate 2 (IRS-2) As a Major Locus for Early- Onset Autosomal Dominant Type 2 Diabetes.  Diabetes.  March 1998,  vl 48.

    12. Authors: Engen, John R, Superti-Furga, Giulio. 17 July 2000.

    13.  Paz, Keren, Hedva Voliovitch, Yaron R. Hadari, Charles T. Roberts, Derek Lel, and Yehiel Zick.  Interaction Between the Insulin Receptor and its Downstream Effectors.  Journal of Biological Chemistry.  March 1996,  vl 271,  pp 6998-7003.

    14.  Lipes, Araki, Patti Bruning, Haag Johnson, and RS Kahn.  Alternative Pathway of Insulin Signaling in Mice with Targeted Disruption of the IRS-1 Gene.  Nature.  November 1994,  vl 372,  pp 186-190.
     

     
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