Dr S M

BHATT

Receptor Tyrosine kinases

Receptor Tyrosine kinases

 Receptors for many growth factors, cytokines, and hormones.

 Binding of signal initiates a kinase cascade, beginning with

autophosphorylation.
Receptor Tyrosine kinases

 Activated receptor is a dimer.

Monomer that dimerizes (e.g., epidermal Starts out as dimer (e.g., insulin
growth factor receptor) receptor)
Receptor Tyrosine kinases

 Autophosphorylation results

Ligand binding causes activation of the

dimer (turns on kinase activity).
 Receptor Tyrosine Kinases #1:
Growth Factor Receptor

Epidermal growth factor stimulates cell

growth, proliferation, and differentiation
by binding to its receptor, which is an
RTK.
 Receptor Tyrosine Kinases #1:
Growth Factor Receptor

 This pathway is an excellent example of signal

amplication.
Growth Factor Signaling
 Receptor Tyrosine Kinases #1:
Growth Factor Receptor

Epidermal growth factor stimulates cell

growth, proliferation, and differentiation
by binding to its receptor.

Signal binding to the receptor leads to

a kinase cascade.

http://www.youtube.com/watch?v=OvvXgzf58MQ
Receptor Tyrosine Kinase #1: Epidermal
Growth Factor Receptor

 Ligand binding leads to

receptor dimerization,
autophosphorylation, and
recruitment of adapter
molecules:
GRB2
Sos
 Binding of adapter molecules
to receptor recruits and
activates Ras (GTP switch).
Receptor Tyrosine Kinase #1: Epidermal
Growth Factor Receptor

•Activated Ras recruits Raf.

•Binding to Ras activates Raf.

•Raf phosphorylates MEK.

•MEK phosphorylates MAP kinase (aka

ERK).

•MAP kinase/ERK phosphorylates its

targets.
Receptor Tyrosine Kinase (RTK)/

Ras GTPase/MAP kinase (MAPK) signaling pathway

Raf

MEK

ERK
Receptor Tyrosine Kinase #1: Epidermal
Growth Factor Receptor

•Activated ERK has many

substrates in the cytosol [e.g.
cytoskeletal proteins,
phospholipase A2, signalling
proteins, and activation of
transcription proteins (STATs).

•ERK can also enter the nucleus to control gene expression by

phosphorylating transcription factors such as Elk-1, growth-factor-
receptor-binding protein 2, and SRF, serum response factor.
Regulation of GTP-Switch Proteins

Guanine nucleotide
Exchange Factor

GTPase Activating
Protein
Regulator of G-
protein Signaling
Ras: a GTP-Switch Protein

https://www.youtube.com/watch?v=NL3ndoSzFo4
RTK Example 2: Insulin Receptor

•Insulin receptor is
already a dimer

•Insulin binding triggers

conformational change
and autophosphorylation
of Tyr residues in the
cytosolic region

•Receptor then binds and

phosphorylates target
proteins
Fig. 12-15 RTK Example 2: Insulin Receptor
•One target is IRS-1.

•Activated IRS-1 recruits Grb2.

•Grb2 binds Sos.

•Sos binds Ras, leading to

nucleotide exchange.

•Ras activates Raf-1.

•Raf-1 phosphorylates MEK.

•MEK phosphorylates ERK/

MAPK, which phosphorylates
transcription factors.
Fig. 12-16 RTK Example 2: Insulin Receptor

•Insulin signaling has many effects. Here’s another one.

b-Pancreatic Islet Cells are the Source of Insulin
Pancreatic islet b-cells with insulin

a-cells with glucagon

 How Relatively Stable Blood Glucose Concentration is
Maintained ?
After a meal Before a meal

Liver
Hepatocytes
The idealized diagram shows the fluctuation of blood sugar (red) and the sugar-lowering
hormone insulin (blue) in humans during the course of a day containing three meals. In
addition, the effect of a sugar-rich versus a starch-rich meal is highlighted.
Insulin Actions

Fast actions

Slow actions
Human Proinsulin
Insulin undergoes extensive
posttranslational
modification along the
production pathway.
Production and secretion
are largely independent;
prepared insulin is stored
awaiting secretion. Both C-
peptide and mature insulin
are biologically active. Cell
components and proteins in
this image are not to scale.
Computer-generated image of six insulin molecules
assembled in a hexamer, highlighting the threefold
symmetry, the zinc ions holding it together, and the
histidine residues involved in zinc binding. Insulin is
stored in the body as a hexamer, while the active form
is the monomer.
Insulin release from pancreas oscillates with a
period of 3–6 minutes
Structure of Insulin Receptor

Insulin binding
domain

Transmembrane
domain

Intracellular
tyrosine kinase
domain
Pierre De Meyts and Jonathan Whittaker
Insulin-mediated glucose transport signaling pathway

Insulin

IR
a a
b b

P
IRS
PI3K

Glut4 Akt
P
Insulin-mediated glucose transport signaling pathway
Glucose Uptake Assay
Insulin

IR
glucose a a
b b

P
IRS
PI3K

Akt
P
GLUT4 translocation
Insulin-mediated glucose transport signaling pathway
Insulin

IR
a a
b b

P
IRS
PI3K

Glut4 Akt
P
Insulin-mediated glucose transport signaling pathway
Glucose Uptake Assay
Insulin

IR
glucose a a
b b

P
IRS
PI3K

Akt
P
 Glucose uptake assay
Insulin-mediated signaling pathway
Insulin
2 deoxy-D-[3H]-glucose
IR
a a
glucose
b b

IRS P
PI3K

Glut4 Akt
P
Glucose transporters
Structural model of the major insulin-responsive glucose
transporter GLUT4
Signal transduction in insulin action
Alternative (supplementary) glucose transport pathway
Current known insulin receptor mediated signaling pathways
Three steps that provide functional divergence for insulin signaling
PI-3 Kinase

1. Type 1A PI-3K in adipocytes

2. Heterodimer – p85 regulatory subunit and a p110 catalytic
subunit
3. IRS1 and/or IRS2 bind PI-3K via SH-2 domain on p85,
which then activates p110
4. 6 isoforms of p85 regulatory subunits – tissue specific
expression
5. Function of PI-3K is to convert phosphotidylinositol 4,5-
bisphosphate (PIP2) to PI 3,4,5-triphosphate (PIP3)
6. Activates three Ser/Thr kinases – Akt/PKB, atypical PKC
isoforms and phosphoinositide-dependent kinases (PDK-1
and PDK-2)
Three steps that provide functional divergence
for insulin signaling
Akt / PKB (Protein kinase B)

1. 3 isoforms, Akt-2 is most highly expressed in adipocytes

2. Phosphorylation and activation of Akt involve 3’ phosphoinositides
and the action of PDK
3. Contains PH domain
4. The downstream targets of Akt/PTB have not been identified.
5. Required for insulin-mediated glucose transport
6. Involved in multiple other insulin responses
7. Represents an important mechanism for “cross-talk” between PI-3K
pathway and other pathways regulating gene transcription and
mitogenic effects.
Negative regulations of insulin signaling
 What is Insulin Resistance?

1. Impaired insulin-mediated glucose uptake in

peripheral cells

2. Impaired suppression of gluconeogenesis by the

liver

3. Impaired suppression of lipolysis in adipocytes

 Summary

Insulin receptor (IR) is a pre-formed tetrameric transmembrane

protein with two a- and two b- subunits linked by disulfide
bonds.
Extracellular a-subunits of IR form the insulin binding site and
intracellular b subunits possess tyrosine kinase activity
Insulin binding to the a subunits of IR triggers a
conformational change in the b subunits and activates the
tyrosine kinase activity of the b subunits. Each b-subunit
trans-phosphorylates Tyr residues on the other b-subunit
(autophosphorylation).
Activated IR phosphorylates insulin receptor substrate
proteins (IRSs)
 Summary II

Phosphorylated tyrosine on IRS serves as docking site for PI-

3K, and moves PI-3K from cytosol to the inner side of plasma
membrane
PI-3K converts PIP2 into PIP3 by phosphorylation
PIP3 moves Akt (PKB) next to membrane bound protein
kinases and protein kinases phosphorylates and activates Akt
Akt indirectly activates GLUT4, inducing the translocation of
GLUT4 from cytoplasm to the plasma membrane and
triggering transport of glucose into the adipocyte (also
muscle) cells
Summary III (Common themes in signal transduction)

Tyrosine kinase receptors for most growth factors

Docking (often SH2 domain to phosphorylated Tyr) for protein-
protein interaction near receptor
Multiple protein factors (often kinases) for signal amplification
and potential crosstalk with other regulatory pathways
May have alternative pathway(s) for further fine tuning for the
signal
Signal inhibitors and attenuators such as phosphatases and/or
serine/threonine kinases coexist with activators
Temporal and spatial regulation of signal