Lecture 21: Cell Signaling Flashcards
examples of cellular signals
growth factors, cytokines, hormones
Signals are received by ______ where?
receptors
at the cell surface (or inside cell)
signals are transduced to where? and do what?
to the nucleus
they change the activity of transcription factors
Changes in gene expression can lead to a wide variety of responses including ____
progression or halting of cell cycle
modification of metabolic pathways differentiation
changes in cell shape or movement.
Some receptors can transduce signals through _____
more than one pathway
Many genes are regulated by multiple transcription factors each of which can be activated by ______
more than one type of signal
There can be ____ between signaling pathways
crosstalk
signal transduction (def.)
process of converting signal into cellular response
Steps in signal transduction
- release or exposure of signaling molecule by signaling cell
- binding of signal to receptor (on target cell)
- Initiation of intracellular signal transduction pathway by activated receptor
- Specific change in cellular function, metabolism or development
- Removal of signal
examples of effector proteins + effect
-caspase -> death
-metabolic enzyme -> altered metabolism
-transcription regulatory protein -> altered gene expression
-cytoskeletal protein -> altered shape or movement
Four types of extracellular signaling
1) contact dependent -> signaling cell binds to receptor
2) paracrine -> close signaling cell
3) synaptic
4) endocrine -> hormone over long distance
contact dependent signaling example
Apoptosis (FasL/Fas)
Paracrine signaling example
Interferon response
Endocrine signaling example
Insulin
Cellular responses to signals can be _____
very fast or slow
______copies of a specific cell surface receptor/cell
1,000-50,000
Receptors comprise _____ of plasma membrane proteins
< 0.01%
Most receptors are_____ that require _____for extraction from cellular material
transmembrane proteins
detergents
Two ways to isolate and identify receptors
1) Affinity purification/chromatography
2) Expression cloning
Affinity purification/chromatography requires ______
highly purified ligand
Expression cloning doesn’t require ___
highly purified ligand
Affinity purification/chromatography steps
1) attach ligand to inert matrix
2) incubate with solubilized membranes (with receptors and other proteins)
3) wash away unbound proteins
4) elute bound proteins with excess ligand
5) identify all proteins in fractions (receptor will be in fraction with ligand)
expression cloning is used for receptors that can’t be _____ or ______
isolated by chromatography
are not abundant enough for direct characterization
expression cloning steps
1) isolate mRNA from cells that express the receptor
2) make cDNA from mRNA
3)insert cDNA into expression plasmid
4) transfect plasmid into cells that don’t express the receptor
4) select cells that have plasmid (ex. antibiotic)
5) identify cells with receptor on cell surface with fluorescent ligand
6) sequence cDNA insert to identify receptor
3 classes of receptors
1) ion-channel coupled receptors
2) G-protein coupled receptors
3) enzyme-coupled receptors
GPCRs stands for ____
G protein-coupled receptors
GPCRs regulate ______
vision, taste, smell, immune system, nervous system, metastasis
Components of GPCR pathway
1) A trans-membrane receptor
2) A hetero-trimeric G-protein
3) An effector regulated by the “activated” G-protein
4) Production of second messengers (cAMP, cGMP, DAG)
5) Second messengers act on downstream target(s) to yield response
G proteins are ____ with subunits named ____
-Heterotrimeric GTP binding proteins
a,b, and g
a-subunit of G proteins does what?
binds and hydrolyzes GTP
___ subunits of G protein are ____ allowing for membrane association
a & g
lipidated
a-subunit of G-protein bound to GTP is ___
on
a-subunit of G-protein bound to GDP is ___
off
when a-subunit of G protein is active/GTP-bound , ____
it dissociates from B and g subunits
There are several ______ of recognizing different receptors and effectors
a, b, and g subunits
GPCRs steps
1) Binding of hormone= conformational change in receptor
2)activated receptor binds to Ga subunit of inactive/GDP trimeric G protein
3) receptor causes conformational change in Ga = dissociation of GDP
4) GTP binds to Ga = dissociation of Ga from receptor and Gbg
5) Hormone dissociates from receptor from receptor, Ga-GTP binds to effector =activation
6) Hydrolysis of GTP = Ga dissociate from effector and re-associate with Gbg
FRET stands for ____
Fluorescence (or Förster) resonance energy transfer
_______ can be used to study interaction between proteins in real time
Loss or gain of FRET
In order to have FRET, you need two things?
1) Spectrum of Em (donor)-emission overlaps with Ex -excitation (acceptor)
b) The two molecules need to be closed
in distance
Using FRET to use to study GPCR pathway
1) Measure extent of fluorescence resonance energy transfer (FRET) before and after addition of ligand
2) Loss of FRET signal occurs very
soon after ligand addition
If ligands aren’t close together, what happens to fret?
you lose FRET
If ligands are close together, what happens to fret?
you gain FRET
RTKs stands for ___
Receptor Tyrosine Kinases
EGF stands for ___
Epidermal growth factor
EGFR stands for ___
Epidermal growth factor receptor
EGFR is what type of receptor?
RTKs
EGFR binds ___ and then activate _____
growth factors
cellular proliferation pathways
EGFR is important in ___
human cancer
EGFR/Her2 phenotype in cancer cells
overproduction of EGFR or always active EGFR
RTK and cancer therapy
anti-RTK therapies
RTK cytoplasmic domains have what type of activity?
intrinsic kinase activity
RTKs all have this domain?
tyrosine kinase domain
in the absence of ligand, RTK has ____
low kinase activity
ligand binding on RTK leads to ____
trans-auto phosphorylation of receptor cytoplasmic domain
the trans-auto phosphorylation generates ____
binding sites for signaling proteins which rely signal downstream
activated RTK steps
1) adaptor protein binds to activated RTK
2) Ras-GEF protein binds to adaptor protein
3) GEF activates Ras protein
4) Downstream signals
____ domains on adaptor protein binds to ____ on RTK cytoplasmic domain
SH2/PTB
phosphotyrosine residues
____ domains on adaptor proteins binds to ______ on Ras-GEF proteins
SH3
proline rich sequences (PXXP)
GEF protein turns Ras protein from __ to __ -bound
GDP
GTP
example of adaptor protein
Grb2
example of Ras-GEF
Sos
_____ (active) signals downstream through additional effectors
GTP-Ras
Ras can signal through ____
MAP kinase pathways
Ras protein is associated with ___
PM
active Ras activates ___ which uses ___ to activate ____ which uses ___ to activate ___
MAP kinase kinase kinase
ATP
MAP kinase kinase
ATP
MAP kinase
MAP KKK (ex.)
Raf
MAP KK (ex.)
Mek
MAP K (ex.)
Erk
Raf (MAP KKK) is recruited to membrane by ___
activated Ras
Hydrolysis of GTP by Ras releases ____ which phosphorylates and activates _____
active Raf (MAP KKK)
MEK (MAP KK)
MEK (MAP KK) phosphorylates and activates _____
MAP K
active MAP K does what?
translocates to nucleus and phosphorylates transcription factors -> changing gene expression
Evidence that Ras functions downstream of RTKs:
1) Microinjection of neutralizing antibodies to Ras blocks proliferation of cells stimulated with growth factors (EGF)
2) Expression of constitutively active Ras
mutant causes cells to proliferate in absence of growth factors
anti-RTK monoclonal antibodies can do what?
block ligand binding or dimerization
Drosophila eye is composed of many ____
Each _____ is composed of _____
The _____ gene encodes an ___ that is
required for _____
ommatidia
ommatidium
8 tubular photoreceptors (R1-8)
sevenless
RTK
development of the R7 receptor
wildtype drosophila eye
1) R8 cell makes surface ligand (Boss)
2) R7 receptor/RTK (Sev) binds Boss
3) R7 precursor with RTK becomes R7 neuron
mutant with no Sev drosophila eye
1) R8 cell makes surface ligand (Boss)
2) no signal transduction in R7 cell
3) R7 precursor without RTK becomes cone cell
mutant with no Sev but always active Ras drosophila eye
1) R8 cell makes surface ligand (Boss)
2) always active Ras -> signal transduction
3) R7 precursor without RTK becomes R7 neuron
Crosstalk between signaling pathways allows cells to _____
differentially transmit signals from cell surface
