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Down-regulation of Phosphoinositide-activated
Second Messenger System
Off-signals:
1. IP3 rapidly dephosphorylated by phosphatases.
2. DAG rapidly hydrolyzed.
3. Ca2+ rapidly pumped out.
4. Ser/Thr phosphatases dephosphorylate PKC and CaM kinase targets.
Fertilization of an Egg by a Sperm Triggering an
Increase in Cytosolic Ca2+
Prior to fertilization, the starfish egg was injected with a Ca2+-sensitive fluorescent
dye. A wave of cytosolic Ca2+ (red), released from the ER, is seen to sweep across
the egg from the site of sperm entry.
Wave of Ca2+ release and PKC activation spreading from the site of artificial
activation of a Xenopus egg.
The egg was injected with calcium red (a fluorescent dye sensitive to the [Ca2+])
and a fusion protein, consisting of green fluorescent protein and PKC, which
produces green fluorescence when PKC is activated.
Protease-Activated Receptors (PARs):
GPCR’s activated by a novel proteolytic
mechanism: Protease forms a Michaelis
complex with its receptor/substrate (E•S) to
proteolytically cleave a small peptide from
the NH2-terminus generating a “NEW”
NH2-terminus
This “new” NH2-terminus serves as a
tethered peptide ligand, binding intramolecularly to the body of the “receptor” to effect
transmembrane signaling.
Proteases are typically serine proteases.
Platelet Thrombin receptor structure: PAR1
Mechanisms of Platelet Activation and Control, Plenum Press, 1993
Features of PAR Amino-terminal Exodomains
PARs’ hirudin-like
sequences
Hirudin
PAR1
PAR3
PAR4
PAR2
C-tail
38
35
43
33
L-DPR
L-PIK
LPAPR
S-KGR
//
//
//
//
DFEEIPEEYLQ
SFLLR-NPNDKYEPFWEDEEK
TFRGA-PPN-SFEEFPFSALE
GYPGQVCANDSDTLELPDSSR
SLIGK-VDGTSHVTGKGVTV-
 Tethered
ligand domains
Cleavage site
61
57
68
55
Cleavage of the receptor/substrate
by the protease results in
“Irreversible” activation
Irreversible Activation by Cleavage
“Reversible” activation can be
accomplished with a small
peptide mimicking the new
NH2-terminus
Reversible Activation by Peptide Mimetic
One Receptor Can Couple to More than One G protein
Four different receptors (R1-4) are shown.
R3 couples to both Gi and Gq
Consensus Sequences for Protein Kinases
Modulation of Glycogen Synthase Activity by Phosphorylation
This enzyme contains nine separate sites in five designated regions susceptible to phosphorylation by
several different cellular protein kinases. Consequently, the activity of this enzyme can be modulated in
response to a variety of second messengers produced in response to different extracellular signals.
Therefore regulation is a matter of finely tuned modulation of the enzyme activity over a wide range and
not merely on/off switching.
Structural/functional Consequences of Lipid Modifications
TIBS 16:338, 1991
• Part of the GPCR C-terminus is tethered to the membrane by palmitoylation through a thioester linkage to a conserved Cys.
• The G protein  subunit has myristate attached via an amide linkage to an N-terminal Gly to effect membrane association.
• The G protein  subunit is incorporated into the membrane via a geranylgeranyl moiety attached via a thioether linkage to
a C-terminal conserved Cys.
• Some G protein-coupled effectors are transmembrane proteins (e.g. ion channels and adenylyl cyclase) as shown here,
others (e.g. phospholipase C) may be peripheral membrane proteins.
Sites of Lipid Modification of G Protein Subunits
TIBS 16:338, 1991
Production of the 2nd Messenger – cGMP
The membrane-bound form
of guanylate cyclase is activated directly by hormone
binding to its receptor, e.g.
atrial natriuretic peptides.
The cytosolic form is a different
protein and is activated by nitric
oxide (NO).
Protein kinase G (PKG) is activated by cGMP.
Functional Domains of the ANF Receptor
Figure 21.44 – Devlin, Textbook of Biochemistry
I. Cell Signaling by Nitric Oxide (NO)
Most cells can produce NO; however, it appears to function primarily in three
broad categories:
1. via the endothelial cell to cause vascular (smooth muscle cell) relaxation;
2. during neurotransmission to facilitate CNS function;
3. in cell-mediated immune responses to facilitate immunologic function.
NO is produced via the enzyme complex, NO synthase (NOS), which deaminates
Arg to form citrulline and NO
II. Cell Signalling by NO
Basal production of NO required to control vascular tone and normal blood pressure.
NO is chemically unstable and decomposes in seconds!!!
NO-mediated intercellular communication:
Agonists such as acetylcholine, thrombin, histamine and bradykinin activate their various
receptors on vascular endothelial cells, leading to activation of NO synthase.
NO diffuses from the EC to the
smooth muscle cell (SMC)
Within the SMC, NO stimulates
production of cGMP via activation
of a soluble guanylate cyclase.
cGMP activates cGMP-dependent
kinases (PKG) with the end result
being SMC relaxation.
Nitroglycerin and nitroprusside,
well known for treating angina
pectoris function by producing
NO during their metabolism.
Second Messengers Involved in Intracellular Signaling
© 2000 by W. H. Freeman and Company. All rights reserved.
Eukaryotic Signal Transduction Systems Involving
Membrane Receptors (1-5) and Second Messengers (1-4)
Second Messengers Involved in Intracellular Signaling
© 2000 by W. H. Freeman and Company. All rights reserved.
Signaling via Enzyme-linked Cell Surface Receptors
Receptor Protein Tyrosine Kinases (RPTKs)
Receptors are characterized by : 1) a single membrane-spanning domain; 2) a large glycosylated
extracellular domain defining the ligand binding site; and 3) an intrinsic enzymatic activity
contained within the cytoplasmic domain (tyrosine kinase activity)
Families of Ligands for Enzyme-linked Receptors
Molecular Basis of Medical Cell Biology, 1998
Ligands for RPTKs
BASIC PRINCIPLES OF RPTK SIGNALING
Ligand-induced RPTK Dimerization and Subsequent Phosphorylation
© 2000 by W. H. Freeman
and Company. All rights
reserved.
Some ligands are monomeric, e.g. EGF. Ligand binding induces a conformational
change in the receptors  receptor dimerization.
Other ligands are dimeric and bring two receptors together directly.
Irrespective, subsequent to dimerization, the kinase activity of each subunit crossphosphorylates Tyr residues near the active site in the other subunit. Autophosphorylation of Tyr residues within other parts of the cytoplasmic domain ensues.
BASIC PRINCIPLES OF RPTK SIGNALING
Function of Phosphorylated Tyrosines
© 2000 by W. H. Freeman and Company. All rights reserved.
Phosphotyrosines of dimerized receptor serve as high affinity binding sites
for “docking” and/or “activation” of intracellular signalling proteins.
BASIC PRINCIPLES OF RPTK SIGNALING
Selected Intracellular Substrates and Targets of RPTKs
Proteins that bind to Tyr-PO4 residues must contain
SH2 or SH3 domains [Src(soluble cytoplasmic tyrosine kinase)
Homology domains]
• SH2 - 1– aa stretch; SH3- 60 aa stretch
• Crystallographic studies demonstrated that these domains
have a pocket into which a Tyr-PO4 residue would fit
• Adapter proteins link other cytoplasmic proteins to the
activated receptor
Molecular Basis of Medical Cell Biology, 1998
Binding of Various Signal-transducing Molecules
to Different Phosphorylated Tyrosines in the PDGF receptor
**
**denote Tyr residues
that are phosphorylated
***
***the outlined area in
each bound signal transduction molecule represents the SH2 domain
Binding pocket
for a specific
amino acid side
chain
Binding pocket for
phosphotyrosine
The 3-D Structure of an SH2 Domain
The SH2 Domain: A Compact
“Plug-in” Molecule
Phospholipase C is Coupled to Gq
Phospholipase C Binds to Phosphorylated RPTKs
Mechanism of Activation of PLC-1 by EGF or PDGF
(a) Binding of EGF (or PDGF) to its receptor elicits autophosphorylation subsequent to
receptor dimerization. (b) The PLC-1 forms a complex with the EGF (PDGF) phosphorylated
receptor and the tyrosine residues 771, 783 and 1254 are phosphorylated, which (c) activates
PLC-1.. (TIBS 16:297, 1991)
PLC-, -, - Share Regions of Significant Homology (X and Y)
(G protein activated)
(Tyrosine-kinase activated)
(TIBS 16:297, 1991)
(TIBS 16:297, 1991)
TIBS 16:297, 1991
How Do Growth Factor Receptors Lead to Cell Proliferation?
The Activation of Ras by an Activated RPTK
The Grb-2 adaptor protein binds to a specific phosphotyrosine on the receptor
and to the Ras guanine nucleotide exchange factor (GEF), which stimulates Ras
to exchange its bound GDP for GTP
What are Ras proteins and how are they activated?
GEF
Molecular Biology of the Cell, 2002
Ras proteins are monomeric GTPases.
GEFs activate Ras by stimulating it to give up its GDP for GTP. The cytosolic
[GTP] is 10 times > [GDP], so Ras readily binds GTP once GDP has been ejected.
Ras-GAPs inactivate Ras by stimulating its GTPase activity and thus its hydrolysis
of GTP to GDP. The Ras-GAPs maintain most of the Ras protein (95%) in an
unstimulated state.
**
© 2000 by W. H. Freeman and Company. All rights reserved.
© 2000 by W. H. ©Freeman
Company.
All All
rights
2000 by W. and
H. Freeman
and Company.
rights reserved.
reserved.
Conformational Changes Accompanying Ras Activation
**Sos is a GEF specific for Ras
The MAP-kinase Ser/Thr phosphorylation pathway activated by Ras
Raf
MAP; mitogen
activated protein
Mek
Erk
Ras activates MAP-KKK (Raf), which in turn activates MAP-KK (Mek) by phosphorylation of select Ser/Thr residues. Activated Mek activates MAP-K (Erk) in an analogous
manner. Erk in turn phosphorylates a variety of downstream proteins resulting in
changes in gene expression and protein activity causing complex changes in cell behavior.
Growth Factor Activation of its RPTK Leading to Cell Proliferation
Leading to
proliferation
THE INSULIN RECEPTOR IS UNIQUE
Insulin-dependent stimulation of glucose uptake
occurs through a Ras-independent pathway!
IRS binds/stimulates PI-3 kinase, which effects an, as yet,
ill-defined cascade of events  the translocation of additional glucose transporters (GLUT-4) to the cell membrane.
New Engl J Med 341:248, 1999
GLUT-4 Translocation to the Plasma Membrane
Signal Transduction, Oncogenes and Cancer
Tumor cells can express mutationally altered forms or levels of proteins involved in signal
transduction – e.g. altered growth factors, growth factor receptors, G proteins, nuclear
receptors, protein kinases, etc.
Tumor cells may contain a normal signal transduction protein, but in excessive amounts.
The responsible gene is called an Oncogene. Corresponding normal gene is a Protooncogene.
How do protooncogenes become oncogenes?
1. Once a cellular “protooncogene” becomes part
of a retroviral genome, it can undergo mutation,
producing an oncogene, which can transform a
normal cell subsequent to retroviral infection.
2. Spontaneous or chemically-induced (carcinogens)
mutations in a “protooncogene.”
3. E.g., ras genes altered in codon 12,13 or 61 have
been detected in  30% of spontaneous and chemically induced tumors in animals and humans.
Most ras oncogene proteins
lack GTPase activity.
Several mutations that
generate ras oncogenes
are positioned close to the
bound guanine nucleotide.
EGFR and Cancer
Several mechanisms by which the EGFR system
becomes oncogenic:
• autocrine ligand loops
• amplification or overexpression of the EGFR
receptor
• deletions or mutations that render the receptor
constitutively active independent of ligands
Activities of EGFR are mediated
by several signal transduction
pathways. The best characterized
is the Ras/Raf/Mek/Erk pathway,
which leads to gene transcription
resulting principally in cell growth
and proliferation.
**
Most common mutation produces EGFRvIII, which has a
truncated extracellular region with a distorted ligandbinding area. Although this mutant cannot bind ligands,
it is trapped in a partially activated state, unable to undergo
downregulation. [(+) ligand dependence; (-) ligand independence]
EGFR as a Molecular Target for Cancer Therapy
Extracellular
MoAbs to EGFR
MoAbs to EGFRvIII
Anti-EGFRvIII vaccine
Bispecific Abs
Immunotoxin conjugates
Ligand-toxin conjugates
Intracellular
Small molecular inhibitors
of EGFR Tyr kinase
Nuclear
Antisense oligos
Ribozymes
PTKRs and Bi-directional Signaling: Eph receptors and Ephrin ligands
(Dodelet VC and Pasquale EB. Oncogene 19:5614-5619, 2000)
Ephrin ligands are membrane-bound, with membrane
association occuring through a GPI linkage (A) or as
transmembrane proteins (B).
Upon cell-cell contact, Eph receptors and ephrins engage
in a class specific manner (A-A; B-B), which leads to
receptor clustering, receptor activation and receptor
phosphorylation (shown for EphB), which in turn provides
docking sites for SH2 domain-containing signaling proteins.
Upon receptor binding, ephrin-B ligands also become
Phosphorylated on Tyr, via an unidentified associated
tyrosine kinase. It is unknown if SH2 domain-containing
proteins also dock to phosphorylated ephrin-B ligands.
Ephrin/Eph interactions have been shown to be important
in clot retraction mediated by activated platelets.
Receptor Protein Tyrosine Kinases
Tyrosine Kinase-associated Receptors
Families of Ligands for Enzyme-linked Receptors
Molecular Basis of Medical Cell Biology, 1998
Ligands for Tyrosine-kinase
associated receptors
Ligands Activating Tyrosine Kinase-Associated Receptors
Primarily involved in signaling between cells of the immune and hematopoietic systems, and
signaling from cells near a site of inflammation
Tyrosine Kinase-Associated Receptors
Redundant Signaling:
Many members of the cytokine family can
stimulate the same response in a cell
Molecular basis of redundancy involves
sharing of receptor transducing subunits
Receptors composed of two polypeptide chains:
Recognition subunit specific for and binds
cytokine.
Transducing subunit initiates signaling
response.
The Interleukin-3 (IL-3) Receptor
“Common”  subunit transduces
signal, which is shared by the
IL-5 and GM-CSF receptors
Subsequent to ligand binding, the receptor is phosphorylated on select Tyr residues by a special
family of cytoplasmic Tyr kinases: Janus kinases (JAKs) of which there are four members (JAK1,
JAK2, JAK3 and TYK2). These kinases appear to reside in close proximity to receptors on the inner
surface of the membrane. These kinases also phosphorylate signaling molecules containing the
required SH2/3 domains that dock on the receptor.
BASIC PRINCIPLES OF TYROSINE KINASE ASSOCIATED
RECEPTOR SIGNALING I
Subsequent to ligand binding, the receptor is phosphorylated on select Tyr residues
by a special family of cytoplasmic Tyr kinases: Janus kinases (JAKs) of which there
are four members (JAK1, JAK2, JAK3 and TYK2). These kinases appear to reside
in close proximity to receptors on the inner surface of the membrane.
BASIC PRINCIPLES OF TYROSINE KINASE ASSOCIATED
RECEPTOR SIGNALING II
Src Tyr Kinases are Required for MAP Kinase Activation of Gene Transcription
The “Gleevec” Story
STI-571
(Signal Transduction Inhibitor 571)
Causes overactive
white blood cell
division
Chromosome Swap in Chronic Myelogenous Leukemia
The Story of Gleevec (STI-571):
The “Magic Bullet” for the Cure of CML (Chronic Myelogenous Leukemia)
and GIST (Gastrointestinal Stromal Tumor)
STI-571 is specific for the unphosphorylated “inactive” form of the fusion protein Bcr-Abl, a cytoplasmic
Tyr kinase known to be involved in cell proliferation signaling pathways (left). It does not interact with
the Src kinase family (right).
Intracellular Receptors:
Nuclear Receptor Superfamilies of Ligand-activated Transcription Factors
Ligands can be steroid hormones derived from cholesterol
(e.g. cortisol, aldosterone, sex hormones, etc.), and related hormones
(e.g. thyroid hormones, vitamin D3 and retinoic acid, etc.), which
typically circulate in plasma bound to specific transport proteins.
Cellular Accumulation of Ligand
• interact with specific intracellular receptors;
• target cell entry is through passive diffusion; the rate of entry is directly
proportional to the intracellular steroid concentration (autocrine regulation);
• inside the cell, the ligand may a) bind to its specific receptor or b) undergo
metabolism;
• receptor criteria: finite binding capacity; high affinity; specific;
• receptor characteristics: oligomeric, phosphorylated proteins (50,000-200,000
daltons) with three functional domains;
SPECIFICITY ACHIEVED BY THE PRESENCE
OF THE RECEPTOR IN THE TARGET CELL
Intracellular Receptors
Molecular Biology of the Cell, 2002
C-terminus provides unique ligand binding site and sites for protein dimerization.
N-terminus defines region essential for transcriptional activation.
Middle domain (80 residues) contains the DNA binding site; exhibits as much as 50%
homology with other receptors; may associate with proteins that modulate their function.
Formation of a receptor/ligand complex can occur in the cytosol or the nucleus and
typically leads to dimerization.
Receptor Activation Subsequent to Ligand Binding
Estrogen Receptor Binding to DNA
The dimeric receptor protein has two -helical regions that bind to both ends of a symmetrical DNA
sequence (AGGT-CAXXXTGACCT), within the major groove.
The Conserved DNA Binding Domain in Steroid Receptors
Formation of a receptor/ligand complex can occur in the cytosol or the nucleus and
typically leads to dimerization of the occupied receptor.
Dimerized receptor binds to the hormone regulatory element (HRE, 8-15 base pairs)
on the target DNA molecule  changes in gene transcription
Positive and Negative Transcriptional Effects of Steroid Receptors
Binding of a receptor dimer immediately adjacent to a transcription
factor leads to synergistic activation of transcription
Binding of a receptor dimer to a
negative hormone response element may displace a positive
transcription factor.
Protein-protein interaction between receptor
dimer and a positive transcription factor
such as AP 1 may block AP 1/DNA binding
and repress the transcriptional response.
Figure 22.15 – Devlin, Textbook of Biochemistry
In a given cell type, the extent and type of
receptor expressed will define the hormone
sensitivity.
Devlin, Textbook of Biochemistry
Schematic representation of the receptor system and its transformation upon binding
of agonist (hormone H) or antagonist (antihormone AH).
With H binding, the inhibitor, hsp 90, is released (due to a
a conformational change within the receptor) and can interact with
its target DNA leading to a specific effect.
With AH binding, hsp 90 continue to bind to the receptor very
tightly, thus no interaction with DNA can occur.
Steroid Receptors as Drug Targets
Tamoxifen:
anti-estrogen;
effective breast cancer therapy
RU 486:
anti-progesterone;
effective contraceptive
NF-B Proteins: Latent Gene Regulatory Proteins
Pivotal to Most Inflammatory Responses
• Proinflammatory cytokines, such
as TNF-, bind to their specific
membrane receptors and initiate
a pathway that activates NF-B,
normally sequestered in an
inactive state through association
with IB proteins.
• A TNF-/receptor interaction
initiates of pathway that marks
IB for degradation.
• Degradation of IB exposes a
nuclear localization signal on NF-B,
which move into the nucleus.
• Once activated, NF-B turns on
the transcription of > 60 genes
that participate in inflammatory
responses.
How glucocorticoids suppress immune and inflammatory reactions
mediated by cytokines
Tumor necrosis factor (TNF)
binding to its receptor leads to
the ultimate degradation of IB
NF-B stimulates the
ultimate production of
inflammatory cytokines
IB binds to
and inhibits the
nuclear translocation of NF-B.
Glucocorticoid induction of
IB synthesis through GC
binding to its intracellular
receptor and stimulating transcription of the gene.
A glucocorticoid interaction with its receptor results in increasing the transcription
of the protein IB, which binds and inhibits the activity of NF-B, a a transcriptional
activator that stimulates transcription of genes for inflammatory cytokines.