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GPCR-Receptors
1. Dept. of Natural
Sciences
University of St. La
Salle
Bacolod City
2. RECEPTORS
Receptor molecules are proteins with 3 domains: extracellular (cell
surface molecule containing a binding site for the ligand, trans-
membrane, and cytoplasmic domains. Receptors that bind to protein
signal molecules usually have a large extracellular ligand-binding
domain (light green). This domain, together with some of the trans-
membrane segments, binds the protein ligand. Receptors that
recognize small signal molecules e.g. adrenaline, have small
extracellular domains, and the ligand usually binds deep within the
plane of the membrane to a site that is formed by amino acids from
several trans-membrane segments.
3.
4. G-protein-coupled receptors
Ligands: epinephrine, glucagon, serotonin,
vasopressin, ACTH, adenosine, etc,
(mammals); odorant moecules, light, mating
factors (yeast)
Receptors: 7 transmembrane α helices;
cytosolic domain associated with a
membrane-tethered trimeric G-protein
Signal transduction: (1) 2nd messenger
pathways involving cAMP or IP3/DAG, (2)
linked ion channels (3) MAP kinase pathway.
5. G -PROTEINS
G Protein-linked
receptors have
an extracellular
N-terminus and
a cytosolic C-
terminus
separated by 7
transmembrane
α helices
connected by
peptide loops.
One of the extracellular segments has an unique messenger-
binding site. The cytosolic loop between the 5th and 6th α helices
specifically binds a particular G protein. Activated G Proteins bind
to enzymes or other proteins and alter the target protein’s activity.
7. G-Proteins consist of
large heterotrimeric,
and small monomeric
G-Proteins.
The G-protein linked
receptor changes
conformation when a
ligand binds to allow
association of the
heterotrimeric G-
proteins (, , and
subunits) with the
receptor.
Ligand-binding
causes the Gα subunit
to release its bound
GDP, pick up a GTP
and detach from the
complex.
8. Either the GTP-Gα complex, the
Gß- G complex or both bind
target protein(s).
The Gα will remain an activating
messenger until the GTP is
hydrolyzed by the Gα subunit.
The "inactive" GDP-Gα will then
reassociate with the Gß-G
complex to rapidly turn down
this pathway when the original
stimulatory signal is removed.
Some G proteins bind K+ or
Ca+2 ion channels in
neurotransmitters.
Some activate kinases
(enyzmes that phosphorylate).
Some cause either the release
or formation of major 2nd
messengers such as cyclic
AMP (cAMP) and Ca+2 ions.
9. http://highered.mcgraw-
Enzymes hill.com/olc/dl/120107/anim0022.swf
activated by
G proteins
catalyze the
synthesis of
intracellular 2nd
messenger
molecules.
Because each
activated enzyme
generates
many 2nd
messenger
molecules, the
signal is greatly The signal is passed on by the messenger
amplified at this molecules, which bind to target proteins
step in the and other signaling proteins in the cell
pathway. and influence their activity.
10. http://higher
ed.mcgraw-
hill.com/olc/
dl/120069/bi
o07.swf
How 2nd
messengers
work.
(a) The cyclic AMP (cAMP) pathway. An extracellular receptor binds to a signal molecule and,
through a G protein, activates the membrane-bound enzyme, adenylyl cyclase. This enzyme
catalyzes the synthesis of cAMP, which binds to the target protein to initiate the cellular
change. (b) The Ca+2 pathway. An extracellular receptor binds to another signal molecule and,
through another G protein, activates the enzyme phospholipase C. This enzyme stimulates the
production of inositol trisphosphate (IP3), which binds to and opens calcium channels in the
membrane of the ER. Ca+2 is released into the cytoplasm, effecting a change in the cell.
11. cyclic AMP is a 2nd
messenger used by a
major class of G proteins.
cyclic AMP (cAMP) is
generated by adenylyl
cyclase which is embedded
in the plasma membrane
with the enzymatic activity in
the cytoplasm. Adenylyl
cyclase is activated by
binding an activated α
subunit of the Gs G-protein.
Phosphodiesterase
continually degrades cAMP
so in the absence of the
ligand and active G-Protein,
cAMP levels are reduced.
13. Protein kinase A (PKA), a
cAMP-dependent kinase,
is the main intracellular
target of cAMP. In the
target tissue, intracellular
effects such as the
activation of the cAMP
pathway can control a
number of cell functions.
Localization of PKA to
specific regions of the
cell by anchoring
proteins restricts the
effects of cAMP to
particular subcellular
locations. One example is
activation of PKA to
cause the stimulation of
glycogen breakdown.
http://bcs.whfreeman.com/thelife
wire/content/chp15/15020.html
14. Regulation of glycogen metabolism
by cAMP in liver and muscle cells.
(a) An increase in cytosolic cAMP activates PKA, which inhibits glycogen synthesis
directly and promotes glycogen degradation via a protein kinase cascade. At high cAMP,
PKA also phosphorylates an inhibitor of phosphoprotein phosphatase (PP). Binding of
the phosphorylated inhibitor to PP prevents this phosphatase from dephosphorylating
the activated enzymes in the kinase cascade or the inactive glycogen synthase. (b) A
decrease in cAMP inactivates PKA, leading to release of the active form of
phosphoprotein phosphatase. The action of this enzyme promotes glycogen synthesis
and inhibits glycogen degradation.
15. After activation by
cAMP, PKA may move
into the nucleus and
phosphorylate specific
gene regulatory proteins.
Once phosphorylated,
these proteins stimulate
the transcription of a
whole set of target
genes. This type of
signaling pathway
controls many processes
in cells, ranging from
hormone synthesis in
endocrine cells to the
production of proteins
involved in long-term
memory in the brain.
Activated PKA can also
phosphorylate and
thereby regulate other
proteins and enzymes in
the cytosol.
16. Activation of gene
expression following
ligand binding to Gs
protein–coupled
receptors. Receptor
stimulation (1) leads to
activation of PKA (2).
Catalytic subunits of PKA
translocate to the
nucleus (3) and there
phosphorylate and
activate the transcription
factor CREB (4).
Phosphorylated CREB
associates with the co-
activator CBP/P300 (5) to
stimulate various target
genes controlled by the
CRE regulatory element.
17. G proteins couple
receptor activation to
the opening of K+
channels in the plasma
membrane of heart
muscle cells. (A) Binding
of the neurotransmitter
acetylcholine to its G-
protein–linked receptor on
heart muscle cells results
in the dissociation of the G
protein into an activated ß
complex and an activated α
subunit. (B) The activated
ß complex binds to and
opens a K+ channel in the
heart cell plasma
membrane. (C) Inactivation
of the α subunit by
hydrolysis of bound GTP
causes it to reassociate
with the ß complex to form
an inactive G protein,
allowing the K+ channel to
close.
18. Hormone-induced activation and inhibition of adenylyl cyclase in adipose
cells. Ligand binding to Gs-coupled receptors causes activation of adenylyl
cyclase, whereas ligand binding to Gi-coupled receptors causes inhibition of the
enzyme. The G ß subunit in both stimulatory and inhibitory G proteins is identical;
the Gα subunits and their corresponding receptors differ. Ligand-stimulated
formation of active Gα ·GTP complexes occurs by the same mechanism in both Gs
and Gi proteins. However, Gsα ·GTP and Giα ·GTP interact differently with adenylyl
cyclase, so that one stimulates and the other inhibits its catalytic activity.
19. Many G
proteins use
inositol
triphosphate
(IP3) and
diacylglyceral
(DAG) as 2nd
messengers
G Protein-
linked
receptors.
20. Phospholipase C
activates 2 signaling
pathways. PIP2 is hydrolyzed by
activated phospholipase C to yield IP3 and DAG.
IP3 diffuses through the cytosol
and triggers the release of
Ca2+ from the ER by binding to
and opening special Ca2+ channels in the
ER membrane. The large electrochemical
gradient for Ca2+ causes Ca2+ to rush out into the cytosol. Together
with Ca2+, the membrane-bound DAG helps to activate the enzyme
protein kinase C (PKC), which is recruited from the cytosol to the
cytosolic face of the plasma membrane.
21. Integrated regulation
of glycogenolysis
mediated by several
2nd messengers.
(a) Neuronal
stimulation of striated
muscle cells or
epinephrine binding to -
adrenergic
receptors on their
surfaces leads to
increased cytosolic
concentrations of the
second messengers
Ca+2 or cAMP,
respectively. The key
regulatory enzyme,
glycogen
(b) In liver cells, ß-adrenergic stimulation leads to
phosphorylase kinase
increased cytosolic concentrations of cAMP and two other
(GPK), is activated by
second messengers, diacylglycerol (DAG) and inositol
Ca+2 ions and by a
1,4,5-trisphosphate (IP3). Enzymes are marked by white
cAMP dependent boxes. (+) activation of enzyme activity, (-) inhibition.
protein kinase A (PKA).
http://highered.mcgraw-hill.com/olc/dl/120109/bio48.swf
22. Disruption of G Protein signaling causes
several human diseases.
Vibrio cholerae secretes the cholera toxin
which alters salt and fluid in the intestine
normally controlled by hormones that
activate Gs G-Protein to increase cAMP.
The cholera toxin enzymatically changes Gs
so that it is unable to convert GTP to GDP.
Gs can not then be inactivated and cAMP
levels remain high causing intestinal cell to
secrete salt and water. Eventually
dehydration can lead to death (cholera).
24. TRANSFORMING GROWTH FACTOR-ß TGF-ß receptors
activate gene
regulatory proteins
directly at the plasma
membrane. These
receptor serine/
threonine kinases
autophosphorylate
themselves and recruit
and activate
cytoplasmic gene
regulatory proteins
called SMADs. The
SMADs then dissociate
from the receptors and
bind to other SMADs,
and the complexes then
migrate to the nucleus,
where they stimulate
transcription of
specific target genes.
25. TGF-Smad signaling pathway. (1) In some
cells, TGF binds to the type III TGF receptor (RIII),
which presents it to the type II receptor (RII).
(2) In other cells, TGF binds directly to RII, a
constitutively phosphorylated and active kinase.
(3) Ligand-bound RII recruits and phosphorylates
the juxtamembrane segment of the type I receptor
(RI), which does not directly bind TGF. This
releases the inhibition of RI kinase activity that
otherwise is imposed by the segment of RI
between the membrane and kinase domain.
(4) Activated RI then phosphorylates Smad3 or
another R-Smad, causing a conformational
change that unmasks its nuclear-localization
signal (NLS). (5) Two phosphorylated molecules
of Smad3 interact with a co-Smad (Smad4), which
is not phosphorylated, and with importin
(Imp-), forming a large cytosolic complex.
(5-6) After the entire complex translocates into the
nucleus, RanGTP causes dissociation of Imp.
(7) A nuclear transcription factor (e.g., TFE3) then
associates with the Smad3/Smad4 complex,
forming an activation complex that cooperatively
binds in a precise geometry to regulatory
sequences of a target gene. At the bottom is the
activation complex for the gene encoding
plasminogen activator inhibitor (PAI-1).
26. Oncoproteins (e.g., Ski
and SnoN) and I-Smads
(e.g., Smad7) act as
negative regulators of
TGF signaling. TGF
signaling generally
inhibits cell proliferation.
Loss of various
components of the
pathway contributes to
abnormal cell proliferation
and malignancy.
Schematic model of Ski-mediated down-regulation of the response
to TGF stimulation. Ski binds to Smad4 in Smad3/Smad4 or
Smad2/Smad4 signaling complexes and may partially disrupt
interactions between the Smad proteins. Ski also recruits a protein
termed N-CoR that binds directly to mSin3A, which in turn interacts
with histone deacetylase (HDAC), an enzyme that promotes histone
deacetylation. As a result, transcription activation induced by TGF
and mediated by Smad complexes is shut down.
27. Blocking Growth Factors and Receptors
Active growth receptors cause cell division
by activating the MAP kinase pathway, and
keep cells alive by activating PKB.
Turning off growth factor receptors
therefore tends both to stop cells dividing
and to kill them.
The drug trastuzumab is effective in slowing
down the progression of breast cancer
because it prevents a growth factor (RGF)
from binding to its RTK.
This slows cell division and promotes cell
death in cancer cells.
28. CYTOKINE RECEPTORS
Cytokine receptors are associated with cytoplasmic tyrosine
kinases. Binding of a cytokine to its receptors causes the
associated tyrosine kinases (called Janus kinases, or JAKs) to
cross-phosphorylate and activate one another. The activated
kinases then phosphorylate the receptor proteins on tyrosines.
Gene regulatory proteins called STATs (signal transducers and
activators of transcription) present in the cytosol then attach to the
phosphotyrosines on the receptor, and the kinases activate these
proteins. The STATs then dissociate from the receptor proteins,
dimerize and activate the transcription of specific target genes.
29. JAK-STAT signaling pathway.
Following ligand binding to a cytokine
receptor and activation of an associated
JAK kinase, JAK phosphorylates several
tyrosine residues on the receptor’s
cytosolic domain. After an inactive
monomeric STAT transcription factor
binds to a phosphotyrosine in the
receptor, it is phosphorylated by active
JAK. Phosphorylated STATs
spontaneously dissociate from the
receptor and spontaneously dimerize.
Because the STAT homodimer has 2
phosphotyrosine–SH2 domain
interactions, whereas the receptor-STAT
complex is stabilized by only one such
interaction, phosphorylated STATs tend
not to rebind to the receptor. The STAT
dimer, which has two exposed nuclear-
localization signals (NLS), moves into
the nucleus, where it can bind to
promoter sequences and activate
transcription of target genes.
30. Overview of signal-transduction pathways triggered by ligand binding to
the erythropoietin receptor (EpoR), a typical cytokine receptor.
Four major pathways can transduce a signal from the activated,
phosphorylated EpoR-JAK complex. Each pathway ultimately regulates
transcription of different sets of genes. (a) In the most direct pathway, the
transcription factor STAT5 is phosphorylated and activated directly in the
cytosol. (b) Binding of linker proteins (GRB2 or Shc) to an activated EpoR
leads to activation of the Ras–MAP kinase pathway. (c, d) Two
phosphoinositide pathways are triggered by recruitment of phospholipase C
and PI-3 kinase to the membrane following activation of EpoR. Elevated
levels of Ca2 and activated protein kinase B also modulate the activity of
cytosolic proteins that are not involved in control of transcription.
31. Signaling from cytokine receptors is terminated
by the phosphotyrosine phosphatase SHP1 and
several SOCS proteins.
Two mechanisms for terminating signal transduction from the erythropoietin receptor
(EpoR). (a) SHP1, a protein tyrosine phosphatase, is present in an inactive form in unstimulated
cells. Binding of an SH2 domain in SHP1 to a particular phosphotyrosine in the activated receptor
unmasks its phosphatase catalytic site and positions it near the phosphorylated tyrosine in the lip
region of JAK2. Removal of the phosphate from this tyrosine inactivates the JAK kinase. (b) SOCS
proteins, whose expression is induced in erythropoietin-stimulated erythroid cells, inhibit or
permanently terminate signaling over longer time periods. Binding of SOCS to phosphotyrosine
residues on the EpoR or JAK2 blocks binding of other signaling proteins (left). The SOCS box can
also target proteins such as JAK2 for degradation by the ubiquitin proteasome pathway (right).
Similar mechanisms regulate signaling from other cytokine receptors.
32. Studies with mutant mice reveal that both the erythropoietin receptor (EpoR)
and JAK2 are essential for development of RBC. Mice in which both alleles of
the EpoR or JAK2 gene are “knocked out” develop normally until embryonic
day 13, at which time they begin to die of anemia due to the lack of RBC-
mediated transport of oxygen to the fetal organs. The red organ in the wild-
type embryos (/) is the fetal liver, the major site of RBC production at this
developmental stage. The absence of color in the mutant embryos (/) indicates
the absence of RBC containing hemoglobin. Otherwise the mutant embryos
appear normal, indicating that the main function of the EpoR and JAK2 in early
mouse development is to support RBC production.
33.
34. RECEPTOR TYROSINE KINASES
RTKs have a ligand-binding
domain on the exterior, a
single transmembrane
domain, and the enzyme
active site on the
cytoplasmic domain.
The RTK can be comprised
of either one protein or two
proteins: a receptor and a
tyrosine kinase. The
separate nonreceptor
tyrosine kinase has a
cytosolic tail that contains
tyrosine residue targets of
the enzyme activity.
35. Cytokine receptors and receptor tyrosine kinases, transduce
signals via their associated or intrinsic protein tyrosine kinases.
The cytosolic domain of RTKs contains a protein tyrosine kinase catalytic site (1). In
both types of receptor, ligand binding causes a conformational change that promotes
formation of a functional dimeric receptor, bringing together 2 intrinsic or associated
kinases, which then phosphorylate each other on a tyrosine residue in the activation lip
(2). Phosphorylation causes the lip to move out of the kinase catalytic site, thus
allowing ATP or a protein substrate to bind. The activated kinase then phosphorylates
other tyrosine residues in the receptor’s cytosolic domain (3). The resulting
phosphotyrosines function as docking sites for various signal-transduction proteins.
36. The cytosolic domain of cytokine receptors associates with a separate JAK
kinase (1). In both types of receptor, ligand binding causes a conformational
change that promotes formation of a functional dimeric receptor, bringing
together 2 intrinsic or associated kinases, which then phosphorylate each
other on a tyrosine residue in the activation lip (2). Phosphorylation causes the
lip to move out of the kinase catalytic site, thus allowing ATP or a protein
substrate to bind. The activated kinase then phosphorylates other tyrosine
residues in the receptor’s cytosolic domain (3). The resulting phosphotyrosines
function as docking sites for various signal-transduction proteins.
37. Cytosolic proteins with SH2
(purple) or PTB (maroon)
domains can bind to specific
phosphotyrosine residues in
activated RTKs or cytokine
receptors. In some cases, these
signal-transduction proteins
then are phosphorylated by the
receptor’s intrinsic or
associated protein tyrosine
kinase, enhancing their activity.
Certain RTKs and cytokine
receptors utilize multidocking
proteins such as IRS-1 to
increase the number of
signaling proteins that are
recruited and activated.
Subsequent phosphorylation of
Recruitment of signal-transduction
the IRS-1 by receptor kinase
proteins to the cell membrane by
activity creates additional
binding to phosphotyrosine residues in
docking sites for SH2-
activated receptors.
containing signaling proteins.
38. Activation of a receptor tyrosine kinase (RTK) stimulates the assembly of
an intracellular signaling complex that lead to cell growth, proliferation or
differentiation. Binding of a signal molecule to the extracellular domain of
RTK causes 2 receptor molecules to associate into a dimer. Dimer formation
brings the kinase domains of each intracellular receptor tail into contact with
the other; this activates the kinases and enables them to phosphorylate each
other on several tyrosine side chains. Each phosphorylated tyrosine serves as
a specific binding site for a different intracellular signaling protein, which
then helps relay the signal to the cell’s interior.
39. RTKs can activate several signal transduction pathways at
once, including inositol-phospholipid-calcium pathway and the
Ras pathway.
Ras is an intracellular GTPase switch protein that acts
downstream from most RTKs. Like G, Ras cycles between an
inactive GDP-bound form and an active GTP-bound form.
Ras cycling requires the assistance of two proteins, a guanine
nucleotide–exchange factor (GEF) and a GTPase-activating
protein (GAP).
RTKs are linked indirectly to Ras via two proteins: GRB2, an
adapter protein, and Sos, which has GEF activity
The SH2 domain in GRB2 binds to a phosphotyrosine in
activated RTKs, while its two SH3 domains bind Sos, thereby
bringing Sos close to membrane-bound RasGDP and activating
its nucleotide exchange activity.
Binding of Sos to inactive Ras causes a large conformational
change that permits release of GDP and binding of GTP,
forming active Ras. GAP, which accelerates GTP hydrolysis, is
localized near RasGTP by binding to activated RTKs.
40. Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs).
The receptors for epidermal growth factor (EGF) and many other growth factors are RTKs.
The cytosolic adapter protein GRB2 binds to a specific phosphotyrosine on an activated,
ligand-bound receptor and to the cytosolic Sos protein, bringing it near its substrate, the
inactive Ras-GDP. The GEF activity of Sos then promotes formation of active RasGTP. Note
that Ras is tethered to the membrane by a hydrophobic farnesyl anchor.
41. In unstimulated cells,
Ras is in the inactive
form with bound GDP;
binding of a ligand to its
RTK or cytokine receptor
leads to formation of the active RasGTP complex.
(1) Activated Ras triggers the downstream kinase cascade
(2-6), culminating in activation of MAP kinase (MAPK). In
unstimulated cells, binding of the 14-3-3 protein to Raf
stabilizes it in an inactive conformation. Interaction of the
Raf N-terminal regulatory domain with RasGTP relieves
this inhibition, results in dephosphorylation of one of the
serines that bind Raf to 14-3-3, and leads to activation of
Raf kinase activity (2-3). Note that in contrast to many
other protein kinases, activation of Raf does not depend on Kinase cascade that
phosphorylation of the activation lip. After inactive RasGDP transmits signals
dissociates from Raf, it presumably can be reactivated by downstream from activated
signals from activated receptors, thereby recruiting RAS protein to MAP kinase.
additional Raf molecules to the membrane.
42. Induction of gene transcription by
activated MAP kinase. In the
cytosol, MAPK phosphorylates
and activates the kinase p90RSK,
which then moves into the nucleus
and phosphorylates the SRF
transcription factor. After
translocating into the nucleus,
MAPK directly phosphorylates the
transcription factor TCF. Together,
these phosphorylation events
stimulate transcription of genes
(e.g., c-fos) that contain an SRE
sequence in their promoter. By
altering the levels and activities of
transcription factors, MAPK leads
to altered transcription of genes
that are important for the cell
cycle. Genes on 22q11, 1q42, and
19p13 affect the MAPK/ERK
pathway and are associated with
schizophrenia, bipolar syndrome,
and migraines.
43. RAS MUTATIONS, CANCER, AND DRUGS
Mutations that stimulate cell proliferation by making Ras
constantly active are a common feature of cancers.
Like other GTPases, Ras turns itself off by hydrolyzing
its bound GDP. Mutant Ras without GTPase activity is
therefore always in the ON state and will be activating
the pathway that terminates in MAPK and cell division
at all times, even in the absence of a growth factor.
Such mutant forms of Ras are found in about 20% of all
human cancers.
Many compounds can inhibit steps in the MAP/ERK
pathway, and therefore are potential drugs for treating
cancer (e.g. sorafenib - a Raf kinase inhibitor and
R115777- prevents Ras from activating MAPK)
Protein microarray analysis can be used to detect
subtle changes in protein activity in signaling pathways.
44. Yeast and higher eukaryotes contain multiple
MAP kinase pathways that are triggered by
activation of various receptor classes including
G protein–coupled receptors.
Different extracellular signals induce activation
of different MAP kinases, which regulate
diverse cellular processes.
The upstream components of MAP kinase
cascades assemble into large pathway-specific
complexes stabilized by scaffold proteins. This
assures that activation of one pathway by a
particular extracellular signal does not lead to
activation of other pathways containing shared
components.
45. Kinase cascade that
transmits signals
downstream from
mating factor receptors
in S. cerevisiae.
The receptors for yeast α
and mating factors are
coupled to the same trimeric G protein.
Ligand binding leads to activation and
dissociation of the G protein. In the
yeast mating pathway, the dissociated
G activates a protein kinase cascade
analogous to the cascade downstream
of Ras that leads to activation of MAP
kinase. The final component, Fus3, is
functionally equivalent to MAP kinase
(MAPK) in higher eukaryotes.
Association of several kinases with the
Ste5 scaffold contributes to specificity
of the signaling pathway by preventing
phosphorylation of other substrates.
46. Many RTKs and cytokine receptors can initiate the
IP3/DAG signaling pathway by activating phospholipase
C (PLC), a different PLC isoform than the one activated
by G protein–coupled receptors.
Activated RTKs and cytokine receptors can initiate
another phosphoinositide pathway by binding PI-3
kinases, thereby allowing the catalytic subunit access to
its membrane bound phosphoinositide (PI) substrates,
which are phosphorylated at the 3rd position.
The PH domain in various proteins binds to PI 3-
phosphates, forming signaling complexes associated
with the plasma membrane.
Protein kinase B (PKB) becomes partially activated by
binding to PI 3-phosphates. Its full activation requires
phosphorylation by another kinase (PDK1), which also is
recruited to the membrane by binding to PI 3-
phosphates.
47. Activated PKB promotes survival of many cells
by directly inactivating several pro-apoptotic
proteins and down-regulating expression of
others.
Signaling via the PI-3 kinase pathway is
terminated by the PTEN phosphatase, which
hydrolyzes the 3-phosphate in PI 3-phosphates.
Loss of PTEN, a common occurrence in human
tumors, promotes cell survival and
proliferation.
A single RTK or cytokine receptor often
initiates different signaling pathways in
multiple cell types. Different pathways may be
essential in certain cell signaling events but
not in others.
48. Recruitment and activation of protein kinase B (PKB) in PI-3 kinase
pathways. In unstimulated cells, PKB is in the cytosol with its PH
domain bound to the catalytic domain, inhibiting its activity. Hormone
stimulation leads to activation of PI-3 kinase and subsequent formation
of phosphatidylinositol (PI) 3-phosphates.The 3-phosphate groups
serve as docking sites on the plasma membrane for the PH domain of
PKB and another kinase, PDK1. Full activation of PKB requires
phosphorylation both in the activation lip and at the C-terminus by PDK1.
49. The cytosolic domain of Eph
receptors has tyrosine kinase
activity. Within the Eph
receptor family, the receptors
exhibit some 30–70%
homology in their
extracellular domains and
65–90% homology in their
kinase domains. Their
ligands, the ephrins, either
are linked to the membrane
through a hydrophobic GPI
anchor (class A) or are
single-pass transmembrane
proteins (class B). The core
domains of various ephrin
ligands show 30–70 %
homology. Ephrin-B
ligands and their receptors
General structure of EPH can mediate reciprocal
RECEPTORS and their ligands. signaling.
50. Like other RTKs, the INSULIN RECEPTOR has an
extracellular domain that binds the transmitter, and a
cytosolic domain with a tyrosine kinase activity. Unlike
growth factor receptors, it exists as a dimer even in the
absence of its ligand. After a large meal, the activated
receptor translocates glucose across plasma membranes
for conversion to glycogen.
51. RECEPTORS THAT ARE ION-CHANNELS
Ligand-gated ion channels
Ligands: neurotransmitters (acetylcholine,
glutamate), cGNP, physical stimuli (touch,
stretching), IP3 (receptor in ER membrane)
Receptors: 4 or 5 subunits with a homologous
segment in each subunit lining the ion channel
Signal transduction: (1) Localized changes in
mebrane potential due to ion influx, (2)
elevation of cytosolic Ca+2
52. LIGAND-GATED ION CHANNELS
These are mechanically gated channels which often
have cytoplasmic extensions that link the channel to
the cytoskeleton. They open on stimulation to allow
passage of Na+, K+, Cl+ or Ca+2 ions.
53. INTRACELLULAR RECEPTOR PATHWAYS
Nitric Oxide Pathway
Ligands: Nitric Oxide (NO)
Receptor: Cytosolic guanylyl cyclase
Signal transduction: generation of cGMP
Nuclear Receptor Pathways
Ligands: lipophilic molecules including steroid
hormones, thyroxine, retinoids, and fatty acids in
mammals and ecdysone in Drosophila
Receptors: highly conserved DNA-binding domain,
somewhat conserved hormone-binding domain, and
a variable domain; located within nucleus or cytosol
Signal Transduction: Activation of receptor’s
transcription factor activity by ligand binding
54. NITRIC
OXIDE (NO)
PATHWAY
Regulation of contractility of arterial smooth muscle by nitric oxide (NO) and cGMP.
Upon activation by acetylcholine, NO diffuses from the endothelium and activates an
intracellular NO receptor with guanylyl cyclase activity in nearby smooth muscle cells.
The resulting rise in cGMP leads to activation of protein kinase G (PKG), relaxation of the
muscle, and thus vasodilation. The cell-surface receptor for atrial natriuretic factor (ANF)
also has intrinsic guanylyl cyclase activity. Stimulation of this receptor on smooth muscle
cells also leads to increased cGMP and subsequent muscle relaxation.
55. NUCLEAR RECEPTORS
Small molecules
(e.g. steroids and
retinoic acid)
diffuse inside
cell, and
ligand-
receptor
complex
translocate
to the
nucleus.
http://highered.mcgraw-
hill.com/olc/dl/120069/bio06.swf
56.
57. Operational model of the
Wnt signaling pathway.
(a) In the absence of Wnt, the
kinase GSK3 constitutively
phosphorylates -catenin.
Phosphorylated ß-catenin is
degraded and hence does not
accumulate in cells. Axin is a
scaffolding protein that forms
a complex with GSK3, ß-
catenin, and APC, which
facilitates phosphorylation of
ß-catenin by GSK3 by an
estimated factor of >20,000.
The TCF transcription factor
in the nucleus acts as a
repressor of target genes
unless altered by Wnt
signal transduction.
b) Binding of Wnt to its receptor Frizzled (Fz) recruits Dishevelled (Dsh) to the
membrane. Activation of Dsh by Fz inhibits GSK3, permitting unphosphorylated -
catenin to accumulate in the cytosol. After translocation to the nucleus, ß-catenin
may act with TCF to activate target genes or, alternatively cause the export of TCF
from the nucleus and perhaps its activation in cytosol.
58. (b) In the presence of Hh,
inhibition of Smo by Ptc is
relieved. Signaling from Smo
causes hyperphos-
phorylation of Fu and Cos2,
and disassociation of the
Fu/Cos2/Ci complex from
microtubules. This leads to
the stabilization of a full-
length, alternately modified
Ci, which functions as a
transcriptional activator in
conjunction with CREB
binding protein (CBP). The
exact membrane
compartments in which Ptc
and Smo respond to Hh and
function are unknown; Hh
signal causes Ptc to move
from the surface to internal
compartments while Smo
does the opposite.
Operational model of the Hedgehog (Hh) signaling pathway.
(a) In the absence of Hh, Patched (Ptc) protein inhibits Smoothened (Smo) protein by an
unknown mechanism. In the absence of Smo signaling, a complex containing the Fused (Fu),
Costal-2 (Cos2), and Cubitis interuptus (Ci) proteins binds to microtubules. Ci is cleaved in a
process requiring the ubiquitin/ proteasome-related F-box protein Slimb, generating the
fragment Ci75, which functions as a transcriptional repressor.
59. Notch/Delta signaling pathway. The extracellular Binding of Notch to its
subunit of Notch on the responding cell is noncovalently ligand Delta on an
associated with its transmembrane-cytosolic subunit. adjacent signaling cell
(1) first triggers cleavage
of Notch by the
membrane-bound
metalloprotease TACE
(tumor necrosis factor
alpha converting
enzyme), releasing the
extracellular segment
(2). Presenilin 1, an
integral membrane
protein, then catalyzes
an intramembrane
cleavage that releases
the cytosolic segment of
Notch (3). Following
translocation to the
nucleus, this Notch
segment interacts
with several
transcription factors that
act to affect expression
of genes that in turn
influence the
determination of cell fate
during development (4).
60. Stimulation by TNFα or IL-1
induces activation of TAK1 NFKß signaling pathway
kinase (1), leading to activation In resting cells, the dimeric
of the trimeric IKB kinase (2a). transcription factor NFKß, composed of
Ionizing radiation and other p50 and p65, is sequestered in the
stresses can directly activate cytosol, bound to the inhibitor IKB.
IKB kinase by an unknown
mechanism (2b). Following
phosphorylation of IKB by IKB
kinase and binding of E3
ubiquitin ligase (3),
polyubiquitination of IKB (4)
targets it for degradation by
proteasomes (5). The removal of
IKB unmasks the nuclear-
localization signals (NLS) in
both subunits of NFKB, allowing
their translocation to the
nucleus (6). Here NFKB
activates transcription of
numerous target genes (7),
including the gene encoding the
subunit of IKB, which acts to
terminate signaling.
61. The NFKB transcription factor regulates many genes that
permit cells to respond to infection and inflammation. In
unstimulated cells, NFKB is localized to the cytosol,
bound to an inhibitor protein, I-B.
In response to extracellular signals, phosphorylation-
dependent ubiquitination and degradation of IKB in
proteasomes releases active NFKB, which translocates to
the nucleus.
Upon binding to its ligand Delta on the surface of an
adjacent cell, the Notch receptor protein undergoes two
proteolytic cleavages. The released Notch cytosolic
segment then translocates into the nucleus and
modulates gene transcription.
Presenilin 1, which catalyzes the regulated
intramembrane cleavage of Notch, also participates in
the cleavage of amyloid precursor protein (APP) into a
peptide that forms plaques characteristic of Alzheimer’s
disease.
62. Proteolytic cleavage of APP, a neuronal plasma membrane protein.
(Left ) Sequential proteolytic cleavage by α-secretase (1) and -secretase (2)
produces an innocuous membrane-embedded peptide of 26 amino acids.
-Secretase is a complex of several proteins, but the proteolytic site that catalyzes
intramembrane cleavage probably resides within presenilin 1.
(Right) Cleavage in the 1 2 extracellular domain by -secretase (1) followed by
cleavage within the membrane by -secretase generates the 42-residue A42
peptide that has been implicated in formation of amyloid plaques in Alzheimer’s
disease. In both pathways the cytosolic segment of APP is released into the
cytosol, but its function is not known.
63. Osteoclasts initially bind to
bone via integrin-mediated Bone resorption and its
podosomes. The subsequent
activation of an osteoclast regulation
by interaction with
neighboring osteoblasts via
the trimeric membrane
proteins RANKL and RANK
induces cytoskeletal
reorganization, leading to
formation of a specialized
tight seal with bone. The
activated osteoclast
secretes into the extra-
cellular space generated by
this seal a corrosive mixture
of HCl and proteases that
resorbs the bone .
Osteoblasts can suppress
bone resorption by secreting
osteoprotegerin (OPG).
Binding of this decoy
receptor to RANKL blocks
RANKL binding to RANK on
osteoclasts and thus their
activation.
64. Signaling pathways
can be highly
interconnected.
Pathways from G-
protein–linked receptors
via adenylcyclase and
via phospholipase C,
and from enzyme-linked
receptors via
phospholipase C and via
Ras. The protein kinases
in these pathways
phosphorylate many
proteins, including
those belonging to the
other pathways. The
resulting dense network
of regulatory
interconnections is
symbolized by the red
arrows radiating from
each kinase shaded in
yellow.
65.
66. Networking of
ECM, CAMs,
junctional
complexes,
signal
transduction
pathways and
the genetic
machinery:
Facebook, Twitter,
and other social
networking sites
pale in comparison!
67. CANCER and
SIGNAL
TRANSDUCTION
Oncogenes are
called
proto-oncogenes
(genes able to
become
oncogenes when
altered by
mutation to their
cancer-causing
condition).
Shown are the
principal classes
of proto-
oncogenes, with
some typical
representatives
indicated.
68. Detailed knowledge of the signaling pathways involved and
the structure of their constituent proteins will continue to
provide important molecular clues for the design of specific
therapies.
Despite the close structural relationship between different
signaling molecules (e.g., kinases), recent studies suggest
that inhibitors selective for specific subclasses can be
designed.
In many tumors of epithelial origin, the EGF receptor exhibits
constitutive (signal-independent) protein tyrosine kinase
activity, and a specific inhibitor of this kinase (Iressa™) has
proved useful in the treatment of several such cancers.
The extracellular domains of many cell-surface receptors can
now be produced by recombinant DNA techniques and have
potential as therapeutic decoy receptors. Already in use is
such a decoy receptor for TNF-α, which binds excess TNF-α
associated with rheumatoid arthritis and other inflammatory
diseases.
69. Drugs that target other signal-transducing proteins
may be useful in controlling their abnormal activities.
For example, inhibitors of farnesyl transferase
(farnesyl groups anchor Ras to cell membranes) are
being tested as therapeutic agents in cancers caused
by expression of constitutively active Ras proteins.
Detailed structural studies of the interaction between
signal-transducing proteins offer exciting possibilities
for designing new types of highly specific drugs.
For instance, knowledge of the interface between the
Sos and Ras proteins or between Ras and Raf could
provide the basis of a drug that blocks activation of
MAP kinase.
As more signaling pathways become understood at a
molecular level, additional targets for drug
development will undoubtedly emerge.
