INSULIN RECEPTORAND

MECHANISM OF SIGNALING

Presented by Juhi & Priyanka, MSc (P).

Introduction: Insulin Insulin is a peptide hormone produced by β-cells of

the pancreas in response to nutritional stimuli. Main sites of action of Insulin in the body include

Liver, Adipose Tissue, Muscles & Brain. Insulin is secreted by β-cells in response to an

increase in Blood glucose levels. Insulin binds to the insulin receptor and facilitates

uptake of glucose by the cell. Insulin’s interaction with the cell triggers both

metabolic and mitogenic cellular responses. Insulin-like Growth Factor, IGF-1 is a mediator of cell

growth and differentiation.http://www.jbc.org/content/271/48/30625.fulhttp://www.abcam.com/pathways/overview-of-insulin-signaling-pathways l

Introduction: Insulin Receptor

Insulin receptor belongs to a family of receptor-tyrosine kinases (RTKs), which phosphorylate their substrate proteins on tyrosine residues.

The insulin receptor comprises of two subunits: the extracellular α-subunit and the transmembrane β-subunit. The functional receptor exists as a dimer/heterotetrameric complex: α2β2

The α-subunit is contains the ligand binding site, as it is the only subunit identified by affinity labelling protocols.

The β subunit is involved in intracellular signalling.

When insulin interacts with the receptor, dimerisation of the receptor takes place. Due to this, the cytoplasmic domains of the receptor come close together faciliating autophosphorylation and triggering the signal transduction pathway.

Insulin Receptor

Types of Signaling Mechanisms

Insulin's interaction with its cell surface receptor triggers both metabolic and mitogenic cellular responses.

In order to do this, Insulin employs two kinds of pathways: Ras-dependant and Ras-independent.

Ras is a member of a large family of small molecular weight GTP binding proteins.

Many RTKs are known to activate the Ras protein in order to mediate signals.

Ras-dependant Pathway: Upon insulin binding, the Ras protein is phosphorylated leading to activation of Mitogen Activated Protein Kinase (MAPK) pathway which is involved in cellular growth and proliferation.

Ras-independent Pathway: In this case, another pathway known as the PI-3/AKT signaling pathway is activated by a series of events facilitating glucose uptake by the cell.

http://www.ncbi.nlm.nih.gov/books/NBK21659/http://link.springer.com/article/10.1023/A%3A1006819008507#page-1http://www.jbc.org/content/271/48/30625.full

Ras-Independent Pathway:PI-3/AKT Signaling

Proteins involved in PI-3/AKT Pathway

Also known as AKT

Role of Proteins Involved in PI-3/AKT Signaling

Insulin & Insulin Receptor: When blood glucose levels rise, insulin released by the beta-cells of the pancreas binds to the Insulin Receptor on muscle cells and/or adipocytes and facilitated dimerisation of the receptor. As a result of this dimerisation, the receptor is autophosphorylated at specific residues.

IRS: Insulin receptor substrate family are proteins that are primarily activated as a result of autophosphorylation. The Ser/Thr residues of these proteins are known to be phosphorylated. There are two types of IRS: IRS-1 and IRS-2. IRS-1 is known to function in glucose metabolism, while IRS-2 functions in lipid metabolism. However, both of these are recruited by the insulin receptor as well as IGF-R to exert different kinds of effects.

PI-3 Kinase: Phosphatidylinositol kinase is activated by IRS-1 and acts as an adaptor protein. Activated PI-3Ks serve to phosphorylate phosphatidyl inositol on the D3 position of the inositol ring generating PtdIns-3,4,5-P3. These specialised lipids recruit PH-domain containing proteins such as Akt to the plasma membrane.

Protein Kinase B or Akt: Akt helps in translocation of GLUT-4 storage vesicles to the plasma membrane by a series of events leading to activation of Rab protein. As GLUT-4 vesicles fuse with the plasma membrane, no. of receptors increase thereby increasing glucose uptake.

Insulin binding to the receptor leads to a conformational change that induces autophosphorylation, similar to activation of other RTKs. After IRS1 binds to a phosphotyrosine residue through a PTB domain, the activated kinase in the receptor’s cytosolic domain phosphorylates IRS1. One subunit of PI-3 kinase binds to the receptor-bound IRS1 via its SH2 domain, and the other subunit then phosphorylates PI 4,5-bisphosphate and PI 4-phosphate to PI 3,4,5- trisphosphate and PI 3,4-biphosphate, respectively. 

IRS-1

AKT

PI-3K

AKT

The phosphoinositides bind the PH domain of protein kinase B (PKB), thereby recruiting it to the membrane. Two membrane-bound kinases, in turn, phosphorylate membrane-associated PKB (AKT) and activate it.

Activated PKB is released from the membrane and promotes glucose uptake by the GLUT4 transporter and glycogen synthesis. The former effect results from translocation of the GLUT4 glucose transporter from intracellular vesicles to the plasma membrane. The latter effect occurs by PKB-catalyzed phosphorylation of glycogen synthase kinase 3 (GSK3), converting it from its active to inactive form. As a result, GSK3-mediated inhibition of glycogen synthase is relieved, promoting glycogen synthesis.

GLUT-4 Translocation: A key step

In the basal state, a dynamic trafficking process ensures that the bulk of GLUT4 is sequestered into intracellular vesicles resulting in a low level of GLUT4 at the cell surface.

Insulin stimulates GLUT4 translocation to the plasma membrane predominantly by releasing GLUT4 from this specialised intracellular pool resulting in a pronounced increase in glucose uptake.

Key mediators: PI-3K, PtdIns-3,4,5-P3, Atk and Rab proteins. Key evidences: Rat adipocytes were pre-incubated with insulin and [3H]2-deoxy-D-glucose, followed by centrifugation

and measurements. Adipocytes pre-incubated with insulin and wortmannin were exposed to [3H]2-deoxy-D-glucose followed by centrifugation and radioactivity measurements. Glucose uptake was found to decrease significantly upon treatment of cells with wortmannin. Since, wortmannin is a specific inhibitor of PI-3k, this study establishes that PI-3k is important for insulin-induced glucose uptake.

Cells were transfected with either wild type (Akt-WT), constitutively active (Akt-myr), or dominant inhibitory (Akt-K179A) forms of Akt, and effects of overexpression of these constructs on insulin-stimulated translocation of a cotransfected epitope-tagged GLUT4 were studied. Overexpression of Akt-WT resulted in significant translocation of GLUT4 to the cell surface even in the absence of insulin. Interestingly, overexpression of Akt-myr resulted in an even larger effect that was independent of insulin. More importantly, overexpression of Akt-K179A (kinase-inactive mutant) significantly inhibited insulin-stimulated translocation of GLUT4. Taken together, our data suggest that Akt is not only capable of stimulating the translocation of GLUT4 but that endogenous Akt is likely to play a significant physiological role in insulin-stimulated glucose uptake in insulin targets such as muscle and adipose tissue.

Many novel proteins have been identified such as PIKfyve and AS160 that have been shown to be involved in GLUT-4 translocated. These are downstream targets of PKB.

Ras-dependent Pathway:Shc/RAS/MAPK Signaling

General Representation of MAPK Signaling: When a peptide hormone/growth factor binds to its receptor and causes subsequent dimerisation, a set of signaling mechanisms are initiated. One such pathway involved mitogen-activated protein kinases (MAPK proteins) that are known to function in cell growth and proliferation. The general mechanism as represented in the figure is similar for all ligands. However, some proteins may differ.

The pathway Insulin binds to its receptor and causes dimerisation. This event facilitates autophosphorylation at the specific tyrosine residues within the

cytoplasmic domain of beta-subunit of the receptor. The phosphorylated tyrosine residues are docking sites for a number of proteins. In

this case primarily two kinds of proteins are activated: Insulin Receptor Substrate (IRS) and Shc family of proteins.

Both these proteins contain the PTB domain that interacts with the phosphorylated residues on the activated receptor. In case of signaling via insulin, both IRS and Shc are important as indicated by this experiment.

A CHO cell line overexpressing the mutant receptor substituting Ala960 for TyrS6O, which was known to show the severely impaired insulin-dependent tyrosine phosphorylation of IRS-1 showed severely reduced insulin-dependent tyrosine phosphorylation of Shc and moderately impaired Ras activation. Using a CHO cell line overexpressing the mutant insulin receptor lacking 82 amino acids of the C terminus of the P-subunit, which was recently reported to exhibit severely impaired receptor kinase activity but retain normal insulin-dependent tyrosine phosphorylation of IRS-1 it was found that insulin induced the submaximal activation of Ras while tyrosine-phosphorylation of Shc protein was severely reduced.

Interference with PTB domains of both IRS and Shc blocks the ability of insulin to elicit cellular responses along both the branches of insulin signaling.

Contd.. IRS proteins contain PH domain that facilitates interaction with specific

plasma membrane sites adjacent to the receptor. Phosphorylated IRS and Shc recruit the SH2 domain containing

proteins such as PI-3K or Grb2 respectively. Grb2 is a growth factor receptor binding protein that also binds the Guanine nucleotide exchange factor, Sos.

Shc-Grb2-Sos activity promotes the dissociation of Ras proteins allowing them to bind to GTP instead. This serves to activate the Ras proteins initiating a cascade of phosphorylation and activation of several Ser/Thr kinases.

https://books.google.co.in/books?id=hgiuDHVUuT4C&pg=PA421&lpg=PA421&dq=ras+dependant+insulin+pathway&source=bl&ots=icjLmRRyDF&sig=k8b2p1ZCghRoIGmoGID6nE8C-SQ&hl=en&sa=X&ved=0CCEQ6AEwAGoVChMIy9m97rv8yAIVhcamCh1MLQwt#v=onepage&q=ras%20dependant%20insulin%20pathway&f=false