1
Endocrine Signaling /
Signal Transduction

•The Insulin Receptor Cascades
Topic 13-2 2
Cell Signaling
Insulin Receptor
IRS-1 P
Y
Y
Y
Y
Y
Y
PI-3
kinase
Insulin
IRS-x P
P
P
MAPK P
P

•The Insulin Receptor Cascades
• Ras
• ras gene - originally discovered as a viral oncogene
•Also part of the normal genome
•Many tumor cells contain a mutant ras
•Protein product Ras
• Small G-protein
• At inner surface of plasma membrane
• Lipid group embedded in inner leaflet
•Forms an important link to nucleus via mitogen
activated protein kinases (MAPK)
Topic 13-2 3
Cell Signaling

•The Insulin Receptor Cascades
• MAPK
•Best characterized group – extracellular signal-
regulated kinase (ERK) family
•Act via either of two pathways:
• RTKs such as the insulin cascade
• Also via G-protein coupled receptors
•ERK activation via coupling to
• Ras and
• Raf a protein serine/threonine kinase
Topic 13-2 4
Cell Signaling

•The Insulin Receptor Cascades
• Ras Raf and MAPK
Topic 13-2 5
Cell Signaling
Insulin

•The Insulin Receptor Cascades
• Ras Raf and MAPK
Topic 13-2 6
Grb-2
Cell Signaling
IRS-1 P
Insulin Receptor
Y
Y
Y
Y
Y
Y
Insulin
P
P
SH2 domain
GTP
test
SOS
(is a GEF)
GDP

• The Insulin Receptor Cascades
• Raf recruitment
• Raf then phosphorylates MAPK/ERK kinase known as MEK
• MEK has dual specificity – phosphorylates
• Threonine
• Tyrosine
• These are separated by one aa residue
• Example ERK2 – Thr 183 and Tyr 185
• Downstream effects of ERK - transcription
Topic 13-2 7
Cell Signaling

•The Insulin Receptor Cascades
•Mechanism of Ras activation
•Mediated by guanine nucleotide exchange factors (GEF)
• Stimulate release of bound GDP
• Exchange for GTP
•Activation of Ras-GTP terminated by GTP hydrolysis
• GTPase-activating proteins interact and accelerate GTP
hydrolysis
Topic 13-2 8
Cell Signaling

•The Insulin Receptor Cascades
•Mechanism of Ras activation
Topic 13-2 9
Cell Signaling

•The Insulin Receptor Cascades
•Ras activation
•A well characterized GEF is Sos
•Sos is bound to Grb2
• Found in the cytosol
•Grb2 is an adaptor protein
• Has no other intrinsic activity
• Has SH2 domain – high affinity for phospho-tyrosine (eg
on IRS)
•Sos-Grb2 localized to the membrane via Ras
Topic 13-2 10
Cell Signaling

•The Insulin Receptor Cascades
•Ras activation
•Sos-Grb2 localized to the membrane via Ras
•Sos stimulates GTP exchange activating Ras
Topic 13-2 11
Cell Signaling

• The Insulin Receptor Cascades
• ERK activation
• As discussed – Raf activates the
MAPK cascade
• ERK translocates to the nucleus
• Regulates transcription factors by
phosphorylation
Topic 13-2 12
Cell Signaling

•The Insulin Receptor
Cascades
•ERK activation
•Many growth factors invoke
rapid transcriptional induction of
immediate-early genes
• Mediated via serum response
element
• Recognized by several factors
including
• SRF
• Elk1
Topic 13-2 13
Cell Signaling

•The Leptin Receptor
Cascades
•Cytokine superfamily
receptors
•No catalytic domain
•Interact with
nonreceptor protein-
tyrosine kinases
• Src family
• JAK family
Topic 13-2 14
Cell Signaling

•The Leptin Receptor Cascades
•JAK/STAT Pathway
•More immediate connection to transcription
•Directly affects transcription factor localization
and function
• Key elements – signal transducers and activators of
transcription (STATs)
•Also other interactions via MAPK and PI3K
Topic 13-2 15
Cell Signaling

•The Leptin Receptor Cascades
•JAK/STAT Pathway
•STATs contain SH2 domains
•Inactive in unstimulated cells
•Localized to cytoplasm
•Leptin binding to its receptor causes recruitment
of STATs which bind to phosphotyrosine via SH2
•STATs are then phosphorylated by Janus Kinase
(JAK)
•STATs then dimerise and are translocated to the
nucleus – stimulation of transcription
Topic 13-2 16
Cell Signaling

•The Leptin Receptor
Cascades
•JAK/STAT Pathway
Topic 13-2 17
Cell Signaling

•The b-adrenoceptor Cascades
•Binding of a small messenger molecule
•Epinephrine/norepinephrine
•Interaction with the receptor causes a
conformational change
•Activates G-protein binding sites in C-terminal
domain
•G-protein interacts with another integral
membrane protein (enzyme) – adenylyl cyclase
•Downstream effects via cyclic-AMP
Topic 13-2 18
Cell Signaling

•The b-adrenoceptor Cascades
Topic 13-2 19
Cell Signaling
- S- S-
Extracellular
domain
Intracellular
domain
Transmembrane
domain

• The b-adrenoceptor Cascades
Topic 13-2 20
Cell Signaling

•The b-adrenoceptor Cascades
• Mechanism of G-protein signaling
•Cyclic AMP an important messenger for a number of
hormone axes
•GTP essential for activation of the enzyme which
generates cyclic AMP – adenylyl cyclase
Topic 13-2 21
Cell Signaling

•The b-adrenoceptor Cascades
• Mechanism of G-protein signaling
Topic 13-2 22
Cell Signaling

•The b-adrenoceptor Cascades
• G-protein structure
•Three subunits – a, b, g
• Heterotrimeric G-proteins
•a subunit binds guanine nucleotides – regulatory
•When inactivated a is bound to GDP and the b and
g subunits
•a and g subunits also attached to membrane via
lipid moieties
Topic 13-2 23
Cell Signaling

•The b-adrenoceptor Cascades
• G-protein function
•Hormone binding induces a conformational change
in the receptor
•C-terminal domain of receptor interacts with G-
protein
• Release of bound GDP
• Exchange for GTP
•Activated a subunit dissociates
•a subunit activates adenylyl cyclase
Topic 13-2 24
Cell Signaling

•The b-adrenoceptor Cascades
• G-protein function
Topic 13-2 25
Cell Signaling

G-protein diversity
•20 different a subunits
•6 b subunits
•12 g subunits
•Different G-proteins associate with different
receptors (c-terminal domain specificity)
•Specific intracellular targets
•b-adrenoceptor-linked G-protein – Gs
•Stimulates adenylyl cyclase
•Other G-proteins inhibit adenylyl cyclase - Gi
Topic 13-2 26
Cell Signaling

•The b-adrenoceptor
Cascades
• Cyclic AMP pathway
•Formed from ATP via
adenylyl cyclase action
•Degraded to AMP by
phosphodiesterase
Topic 13-2 27
Cell Signaling

•The b-adrenoceptor Cascades
• Cyclic AMP pathway
•Most effects of cAMP mediated via protein
kinase A (PKA)
• cAMP binds to regulatory subunits of PKA
• Regulatory and catalytic subunits dissociate
• Catalytic subunits activated
• Phosphorylate serine residues on target proteins
•Good example of signal amplification
Topic 13-2 28
Cell Signaling

•The b-adrenoceptor
Cascades
• Cyclic AMP pathway
•Activation of protein kinase
A
Topic 13-2 29
Cell Signaling

•The b-adrenoceptor Cascades
• Cyclic AMP pathway
•Many cases – activation of transcription of
target genes containing cAMP response element
(CRE)
•Signal carried by catalytic subunit of PKA
• Phosphorylates transcription factor – CRE-binding
protein (CREB) - dimerises
•Activation of cAMP-inducible genes
Topic 13-2 30
Cell Signaling

•The b-adrenoceptor
Cascades
• Cyclic AMP pathway
Topic 13-2 31
Cell Signaling

• Cyclic AMP pathway
•Important in proliferation, differentiation of
many cell types
•Protein kinases
•Protein kinase A
•Not independent action
•Protein phosphorylation reversed by
phosphatases
• Terminate responses following receptor activation of
protein kinases
Topic 13-2 32
Cell Signaling

•Protein kinases
•Protein kinase A
•Level of phosphorylation of substrates finely
controlled by balance of actions of the
kinase and phosphatases
Topic 13-2 33
Cell Signaling

•The Insulin Receptor Cascades – Interaction
with the Leptin Pathway
Topic 13-2 34
Cell Signaling
OB-RlL
OB-Rs
Leptin
MAPK P
Y
Y
Leptin
P P
Insulin Receptor
S
T
A
T
S
T
A
T
Shc
JAK
IRS-1 P
Y
Y
Y
Y
Y
Y
PI-3
kinase
Atypical
PKC
GLUCOSE
FFA
Insulin
INTERSTITIAL
SPACE
IRS-x P
Nucleus
PPAR
Mitochondrion
Fatty Acid Ox-n.
CD36
ACC P
GLUT4
Akt
P
P
P
AMPK
P