biol 303

lecture 20signal transduction II

Alberts ch 16

administrative
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YOU (sophomores): Monday Nov 23
1:30-2:30 UC312
see Bb Course Documents for details.
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remember: online homework

cell signaling
by the end of this (series of) lectures, you should be
able to:
describe the classes of molecules that signal to a cell and
those that enable a cell to respond
compare and contrast the steps in ion channel, G-protein,
and enzyme-coupled receptor signaling pathways
know several examples of receptor signaling pathways in
humans
describe several different effector molecules and their
substrates
describe several second messengers and know in which
pathways they act
describe the types of physical changes that
activate/inactivate signaling molecules
explain how signaling can be amplified or turned off

cells react to various external signals

biological
molecules: proteins,
peptides, aas,
lipids, etc
the external
environment (food,
odor, light, heat,
drugs, pathogens,
etc not shown)

different cell types can respond

differently to the same signal
input: acetylcholine

tputs (phenotype)
olecularly controlled by receptors, second messengers, and effector

generic signal
transduction
cascade
SURFACE receptor gives
information to the cell upon
binding a SIGNAL (LIGAND) by:
generating a RELAY of diffusible
second messenger(s) or by
creating a docking site that other
proteins
willthe
bind
players in
cascade
are reversibly changed,
which alters their
function, mediating
information

signal
receptor
intracellu
lar
signaling
molecule
eff s
ec
to
r
s
outp
ut

response to a signal depends on the

type of receptor to which it binds
RECEPTOR CLASSES:
cell surface/
transmembrane
receptors

G-protein coupled receptors (GPCR)

enzyme coupled receptors (largely
receptor tyrosine kinases)
ligand-gated ion channels
contact-mediated (integrins, Notch/Delta)
intracellular receptors like steroid
hormone receptors (nuclear receptors,
NO)

G-proteins can
activate enzymes
that produce
second
messengers
G-proteins activate (or
inactivate) downstream
proteins
best-characterized:
activation of adenylyl
cyclase to produce cyclic
AMP
cyclic AMP has
widespread effects

warm up question: did you eat

breakfast/lunch today?

A. yes -

your beta cells are

secreting insulin
B. no - your alpha cells
are secreting
glucagon

glucagon
glucagon produced by pancreatic cells
activates a GPCR in liver cells, generating
cAMP and promoting glycogen breakdown

this is an example of what kind of

signaling?
A.
B.
C.
D.

external
autocrine
paracrine
endocrine

glucagon promotes
glycogen breakdown
in liver cells results in
glucose release into
the bloodstream

adrenaline promotes
glycogen breakdown
by a similar signaling
cascade in muscle cells
(for fighting or fleeing)

glucose to the
bloodstream to
feed cells

... but wait, activated PKA can do MORE!

glucagon also promotes

transcription
n liver and other cells

PKA activates the

transcription factor CREB

adrenaline activates
a similar signaling
cascade in muscle cells

insulin
insulin produced by pancreatic cells
activates an RTK in pancreatic beta cells,
regulating their function (we will worry about
how later)

this is an example of what kind of signaling?

A.
B.
C.
D.

external
autocrine
paracrine
endocrine

clicker question - cholera

toxin

bacteria Vibrio cholerae secrete cholera toxin (Ctx)

when infecting the small intestine
results in cholera- diarrhea and rapid dehydration
(50% lethal if untreated)
enters the cell by endocytosis, and A
subunit ribosylates Gs alpha leading
to constitutive activation of adenylyl
cyclase
results in secretion of H2O, K+, Cl-,
what
you
think the critical
Na+,do
HCO
3substrate for PKA phosphorylation is
in this case?
a) ion channels at the membrane
b) CREB in the nucleus
c) cytoskeletal regulators
d) triglyceride lipases

of PKA targets, what response is

slowest?
a) ion channels
b) creb in nucleus
c) cytoskeletal
regulators
d) triglyceride
lipases

different cell types can respond

differently to the same signal
input: acetylcholine

tputs (phenotype)
olecularly controlled by receptors, second messengers, and effector

ion channels can act as

signaling receptors directly

capsaicin/heat
menthol/cold
ligands for ligand-gated channels

what is your favorite ion channel? (Hint: your

favorites are the ones you are using to think
about this question and push the clicker button)

a) Na+
b) K+
c) Ca++
d) all of the above

GPCRs can activate ion channels

heart pacemaker cells
beta/gamma mediated
here the G
protein is in the
Gi class

generic
signal
transduction
cascades
TM receptors act to relay
information into the cell upon
binding a LIGAND either by
generating a diffusible second
messenger
OR by creating a docking site
that other proteins will bind
(OR BOTH)
players in the cascade are
reversibly changed, which
alters their function/activity

activation of receptor
tyrosine kinases (RTKs)

note: cytoplasmic tyrosine kinases also

exist

ligands (upstream)

growth
factors eg,

many growth factors signal this way

include

intracellular phosphorylation of the

TM receptor recruits proteins via Src
Homology Domain 2 (SH2) or

Endothelial Growth Factor

(EGF), Platelet Derived
Growth Factor (PDGF),
Nerve Growth Factor

a variety of signaling proteins

downstream of receptor
phosphorylation

adaptor
transcription
factor
docking
protein
enzymes

many RTKs activate Ras

the Ras family of protooncogenes

monomeric
GTPase!
H-Ras

ras mutations are

found in ~25% of
human cancers;
often constitutively
activated
has GTPase activity
active when
bound to GTP
(inactive when
bound to GDP)
associated with the
membrane by lipids

some signaling molecules

act as switches

(monomeric) G protein
activation cycle
GTPase-activating
proteins (GAPs)
shorten the active
time of the G-protein.
Guanine nucleotideexchange factors
(GEFs) stimulate the
exchange of GDP for
GTP.
constitutively active binds but doesnt
hydrolyze GTP
point mutation!
dominant negative cant bind GTP but
binds factors and sequesters them

Guanine nucleotidedissociation
Ras
r
o
f
F
E
G
inhibitors
Sos is a (GDIs)
inhibit release of GDP.

Ras activates the MitogenActivated Protein (MAP) kinase

cascade
results in activation of
transcription factors or
protein activity
mitogens factors that
promote growth/division

can transmit different types

of information:
Differs in different
cells/situations.
Specificity of the MAP
kinase response due to
differences in the types of
kinases participating and
differences in spatial
organization of

binding of insulin to its receptor

promotes glucose uptake
how?

Autophosphorylated receptor associates

with
insulin receptor substrate proteins

key protein-protein interaction

domains
phospho-Tyr sites are bound by
interacting proteins that contain

SH2
or

PTB
domains.
(in contrast, SH3-domain-containing
proteins bind to proline rich domains
in other proteins)

tYrosine-phosphorylated IRS activates

a variety of signaling pathways
binding site for :

IRS

the role of some molecules

here
is to recruit effectors to the
membrane when the RTK is
activated these are
called adaptors (Grb2)
or docking proteins (IRS)

tyrosine-phosphorylated IRS activates

a variety of signaling pathways

IRS

AKT

PI3K-Akt signaling

phosphoinositide 3-kinase

Akt is a serine/threonine kinase

-promotes growth (through Tor)
-and survival (through Bad/Bcl2)

regulation of glucose uptake in muscle

and fat cells by insulin

Akt

Akt regulates glucose

uptake by GLUT4
transporters.
GLUT4 transporters
reside in intracellular
membrane vesicles.
Vesicles fuse with
the membrane in
response to ligand
binding to the IR.
Diabetes mellitus is
caused by defects in
insulin signaling and

world diabetes day is

saturday
Worldwide diabetes cases hit a new
record at 382 million in 2013,
compared with 371 million cases last
year, according to the latest
estimate by the International
Diabetes Federation.
Researchers say the number of
people living with diabetes may rise
to 640 million by 2040.
The disease currently accounts for
$548 billion in annual health care
spending, and that figure is likely to
reach $627 billion by 2035,
researchers added.

Pathways can work through multiple

downstream components

IR

clicker : you are studying the Sos

protein, which is a Ras GEF

clicker : you are studying the Sos

protein, which is a Ras GEF
in cell culture cells,
you mutate the
Grb2 binding site
in Sos so they no
longer associate.
will the cells have
any response
when you add
insulin?
a) yes
b) no

clicker : you are studying the Sos

protein, which is a Ras GEF
will the sos mutant
cells activate the
MAPK pathway in
response to
insulin?
a) yes
b) no

clicker : you are studying the Sos

protein, which is a Ras GEF
now you make a
fusion protein with the
Grb2 SH2 domain and
all of the Sos protein.
you also delete the
normal Grb2. will cells
activate the MAPK
pathway in response
to insulin?
a) yes
b) no

how do you get a cartoon from

a cell?

del
o
m
n
i
s
s
c
m
i
s
t
i
e
n
gen orga

biochemistry

molecular biology

example: Western blots

genetic analysis + protein interactions

proteininteractions can
be assayed by
immunoprecipita
tion,
pull-down
assays,
mass-spec, etc

phospholipids and sugars are

second messengers

inositol 1,4,5
triphosphate (IP3)
and

diacylglycerol (DAG)
many downstream responses can be
observed in response to these
signaling molecules

IP3 promotes calcium

release from ER
DAG recruits and
activates Protein

calcium wave in a starfish egg due

to fertilization

blue- low [Ca++]

red- high [Ca++]

notice
asymmetry
http://www.youtube.com/watch?v=BH06WgFua_4

Ca2+ is an important second

intracellular messenger
Calcium levels are low (10-7M)
in the cytosol because it is
pumped into the extracellular
space and ER and the
membrane is highly
impermeable to the ion.
Calcium channels can be
transiently opened by action
potential or calcium itself.
Calcium binds to calciumbinding proteins (such as
calmodulin), which affect
other
high
proteins.
Ca2+

Calmodulin is highly conserved and regulates many

signaling molecules in response to Ca2+ binding
+

activates Cam-kinases

this is also one way ligand-gated channels can

act as signaling molecules- widespread cellular
effects when ion concentrations change (TRP)

Pathways Can Be Complicated

convergence of signals transmitted

from a GPCR, an integrin, and receptor
tyrosine kinase

d
un
o
ar
g
tin re?
a
lo he
f
l l y a sm
a
re opl
s
a cyt
R
is t h e
in

crosstalk between
two major signaling pathways
lots of signaling is
occurs
simultaneously
cAMP can block
signals
transmitted
through the MAP
kinase cascade
Ca2+ and cAMP
can influence
each others

convergence, divergence, and crosstalk

among signal transduction pathways

next time:
more signaling, Viagra, and cell
death

a handy table to complete and use as a study

guide
protein

function

pathway(
s)

interacts with
____upstream/signals
to___downstream

other notes

GPCR

many small molecule

ligands/heterotrimeri
c G-proteins then
cAMP or
phospholipids

signal is amplified;
receptors are
rapidly desensitized

TM receptor
and kinase

RTK

many protein
ligands/adaptors,
docking proteins,
TFs, enzymes

when Pled recruits

proteins with SH2 or
PTB domains

adenylyl
cyclase

enzyme/eff
ector

GPCR

Galpha subnits/cAMP
allosterically
activates PKA

PKA

protein
GPCR
kinase/effec
tor

Ras

monomeric
G-protein

PI3K

lipid kinase/
effector

Grb2

adaptor

7-pass TM
(eg, odorant receptor

GPCR

Rs,
rhodopsin,
epinephrine
R)

RTK
(eg, insulin
R, EGFR)

cAMP
activates/Plates
many targets

activates ion
channels, TFs,
cytoskel, metabolic

GTPase activity/
oncogene

another handy table! second messengers

molecul
e

type of
molecule

pathway(
s)

generated
by____upstream/signa
ls to___downstream

DAG

lipid

GPCR

PLC/

IP3

lipid

cAMP

cyclic
nucleotide

other notes

PLC/IP3 receptor
Ca++ release

Ca++
NO

can also act as

a paracrine
factor (from
another cell)

PIP3

binding site of
PH domains (ie,
AKT)

G-proteins can
activate enzymes
that produce
second
messengers
G-proteins activate (or
inactivate) downstream
proteins
best-characterized:
activation of adenylyl
cyclase to produce cyclic
AMP
Galpha switches itself off
by hydrolyzing GTP
(within seconds)
enzyme may stay on or