Transcript Tyrosine kinases can be cytosolic or integral membrane

Lecture 27- Birge
Mechanisms of action of
peptide hormones
Raymond B. Birge, PhD
Learning Objectives
•Review basic biology of Insulin/Glucagon in homeostasis
•Review receptor classifications (emphasis on GPCRs versus
Receptor tyrosine kinases)
•Understand basic biological distinctions between Glucagon
and Insulin
•Understand basic signaling mechanisms between Glucagon
and Insulin
Signaling and Cellular Communication in
Metazoans
600-700 million years ago; origin of multicellular organisms
Examples of Hormones
I. Peptides and Proteins
(Growth factors)
II. Fatty acid derivatives
(Prostaglandins,
Eicosanoids)
III. Amino acid derivatives
(Thyroxine, Epinephrine)
IV. Steroids
(Estrogen, Progesterone)
Examples of second messengers
I. cyclic nucleotides
(cAMP, cGMP)
II. Lipid products
(DAG, phosphoinositol lipids)
III. Ions, small molecules
(Ca2+, NO, Na+, K+)
IV. Modified proteins
(phosphorylation, modular
(domains)
The plasma membrane partitions growth factors and
signaling cascades
Polar
Non-polar
Polar
1. Highly specific
transmembrane
receptor
2. Signal is changed
and amplified
3. Cell responds with
biological readout and
downmodulates
initial signals
Types of growth factor/target cell interactions
1. Paracrine
GF produced by distinct
Insulin/
target cell
Glucagon/NGF
2. Autocrine
GF produced by receptor- EGF/
expressing cell
tumors
3. Juxtacrine
GF produced by distinct
target cell but remains
associated with target cell
TGF-a
4. Holocrine
GF has no signal sequence,
released from target cell by
unknown mechanism
IL’s
Paracrine/Endocrine Growth Factor Signaling
Examples: Insulin, Glucagon
Autocrine Growth Factors
Examples: Oncogenic cells (ie Breast cancer cells
Overproduce EGFR (Her-2) and EGF.
Paracrine Growth factor Signaling
Examples: Neurotrophic fcators (NGF, BDNF, GGF)
Receptor Classification by Mechanism of Action
G Protein-Coupled Receptor (GPCR): 7 transmembrane receptors
(Glucagon, ACTH, TSH, LH, Calcitonin, PTH, Thrombin, Dopamine etc)
Receptors with Intrinsic Enzymatic Activity (Type I or Type II):
(Insulin, EGF, NGF, FGF, VEGF, NT-3, BDNF, etc)
Receptors with Associated Enzymatic Activity:
(Cytokines, EPO, TGF- , BMPs, Interferons)
Receptors that Stimulate Intracellular Proteolysis:
(IL-1, TNF- )
How are signals propagated through cells?
4 Distinct Elements:
1. Signal. Generation, processing,
and release of growth factor
2. Signal Detection. Surface receptor/cytoplasmic receptor
3. Signal Transduction. (PM -> cytoplasm -> nucleus)
4. Alteration in gene expression.
Biology of Insulin and Glucagon
In maintenance of blood sugar
Insulin: Secreted by beta cells of
the pancreas in response to high
blood sugar.
In response to insulin, cells (muscle,
red blood cells, and adipocytes) take
up glucose from the blood.
Glucagon: Secreted by alpha cells of
the pancreas in response to low blood
sugar.
When blood glucose is high, no glucagon
Is secreted
When blood glucose is low, glucagon
causes the liver to release stored
glucose into the blood.
10-10
10-10 M
10-7
cAMP
ATP
PKA
P
phosphorylase
kinase
P
phosphorylase
P
glycogen
synthase
10-6
Signal; ie Insulin
Kinetics of induction of immediate and delayed gene
expression following growth factor stimulation
Figure 6.2 The Biology of Cancer (© Garland Science 2007)
Table 6.1 The Biology of Cancer (© Garland Science 2007)
Figure 6.3 The Biology of Cancer (© Garland Science 2007)
Receptor Tyrosine Kinases
Receptor protein-tyrosine kinases transmit signals across
the plasma membrane, from the cell exterior to the
cytoplasm.
In cells transformed by many oncogenic proteins-(ie, ErbB, EGFR, Bcr-Abl)
there is dramatic increase in protein tyrosine phosphorylations
PROTEIN KINASES
Each kinase phosphorylates particular sequence motifs
Phosphorylation is not restricted to one site on the polypeptide chain
Proteins can be phosphorylated by more than one kinase
(allows convergence of several signaling pathways)
Phosphorylation can effect enzymatic activity
* alter electrostatic interactions and alter equilibrium from one conformation to another
* instrumental in forming new interactions (ie, H-bonding, SH2 domains)
* expose domains or motifs buried in the protein structure (ie NLS and NES)
Consensus amino acids can be used to identify kinase specificity
Polypeptide Growth Factors activate Receptor Tyrosine
kinases (RTK) by a common mechanism
GF-induced activation of a
Receptor Tyrosine Kinase.
1. Ligand-induced dimerization
2. “Auto-phosphorylation”
of the RTK on the cytoplasmic
or intracellular domain.
3. The dimeric state of the receptor
is said to be “activated”
Figure 6.8b The Biology of Cancer (© Garland Science 2007)
The pocket of the SH2
domain is highly specific
for phospho-tyrosine
because of the bulky size
of the tyrosine.
SH2 specificity is
achieved by amino acids
directly C-terminal to the
pTyr (particularly +1, +2,
and +3).
GF-induced activation of a
Receptor Tyrosine Kinase-part 2.
4. Phosphorylation on tyrosine acts
as a “magnet” to recruit cellular
proteins with Src Homology-2 (SH2)
domain.
5. One of these proteins is called
Grb2 (Growth Factor Receptor Binding
protein-clone-2), an intracellular
adaptor protein with SH2 and SH3
domains.
6. Simultaneously, Grb2 binds the
activated RTKAND SOS, an exchange
factor for the G protein, Ras to provide
a link in the signal transduction.
GF-induced activation of a
Receptor Tyrosine Kinase-part 3.
7. Because Grb2 recruits SOS to the
plasma membrane, it meets up with its
partner Ras, resulting in the conversion
of Ras-GDP into Ras-GTP.
8. Ras-GTP is activated and binds more
signaling proteins, leading to the
activation of MAP kinase.
9. Activated MAP kinase is translocated
into the nucleus to activate transcription
factors.
The Ras Cycle
Common Ras mutants
Ras-GDP (Inactive)
Pi
GDP
V12 Ras= Always active
i.e, constitutive activation of
downstream pathways
N17 Ras=Always inactive
i.e, dominant negative for
activation of downstream
pathways
GAP
*
GTP
Ras-GTP (Active)
V12,N17=Functionally-dead
molecule ie, no effect in
downstream pathways
The MAP Kinase (Extracellular-Related Kinase ERK) Cascade
Mitogens, Growth Factors
GTPase
Ras
MAP4K
RA F
MAP3K
MAPKKK
MAP2K
MAPK
Rac, Cdc42
PAK
MAPKK/MEK1
MAPK
Cytokines, Cell Stress
MEKK1
}
Serine/Threonine
Kinases
JNKK/SEK
JNK
Transcriptional Regulation
Cytosol
Nucleus
Many growth factor signals terminate via the
activation of transcription factors
Raf
Localization of MAP K
Summary and Take-Home Points
1. Identify differences between autocrine, paracrine, and
endocrine signaling
2. Distinguish major differences between insulin and
glucagon signaling, and differences in how receptors
are activated (ie, RTK versus GPCR)
3. Understand RTK signal transduction mechanisms
4. Relationship between RTK activation and gene expression