lecture 4 Flashcards
example of enzyme coupled receptors
PDGF; paracrine
what is PDGF molecule structure-wise
dimer
what does PDGF indicate
there’s a wound near by
what is PDGF
a ligand
what are RTKs always activated by
ligand dimer
what are inactive receptors (structure-wise)
monomers
what is the one exception to enzymes activated by ligand dimers
EGF (epithelial growth factor)
describe EGF
signaling molecule isn’t a dimer but still dimerizes receptor complex
describe inactive RTK
enzymatic and kinase activity is off
what happens when ligand binds RTk
binds to 2 RTKs (cuz its a dimer)
what does binding do
brings the inactive receptors in close proximity, initiates kinase activity –> trans autophosphorylation
what kind of activation in PDGF
dimerized induced activation
how are RTKs diff from GPCRs in terms of ligands
RTKs have restricted subset of ligands
what are major class of signaling ligands that bind and activate RTKs
gowth factors
what kind of receptors are growth factors
paracrine receptor
what kind of receptor is Eph receptor
contact-dependent signaling
who has more variety in structure RTK or GPCR
RTK s have more variety
basically how are RTKs activated
2 tyrosine kinase domains are brought together when receptor binds to its dimerized ligand, becomes active, phosphorylate each other
what is initiating step in transmitting signal across plasma membrane
dimerization of receptor in response to ligand binding
what is stage 1 kinda
PDGF binds, brings inactivated receptors close together, triggers minor conformational change in receptor (activates intrinsic kinase activity at low level)
how much kinase activity is activated
just enough that RTK can phosphorylate its neighbor at one position (single phosphorylation on cytoplasmic tails)
what triggers enhancement of kinase activity (step 2)
the single phosphorylation on tail
what is step 2 kinda
much more phosphorylation events happening; both receptors phosphorylated at multiple sites
what does this phosphorylation create
hella scaffolding domains that allow other signaling molecules to bind to receptor
what does allowing other signaling molecules to bind to receptor do
allows them to be at right place at right time to transmit signals deeper and deeper into cytoplasm
what is another thing that allows them to transmit signals
they themselves become activated upon binding
what does dimerization lead to
conformational change which leads to low level activity that does one phosphorylation event on either receptor; that elevates kinase activity even farther, then you get all this phosphorylation going on to bring in more signaling proteins that are activated to relay signal downstream
overall describe autophosphorylation
one phosphorylates the other, both serve as docking sites for downstream signaling proteins
what is exception to the rule
EGF
how is EGF an exception
ligand isn’t dimerized; still it binds and brings together 2 components of EGF receptor
describe EGF receptor
not symmetrical; 2 subunit receptors behave differently
what are the EGF receptor
one is activator other is receiver
what happens when ligand binds activator
triggers conformational change that activates kinase activity of receiver
what does EGF receptor receiver do
phosphorylates itself on long flexible tail, and tail of activator
what is end result of EGF
same; dimerized receptor w/ multiple tyrosine phosphorylations to provide docking sites for signaling molecules
why is EGF a rare case
only one member of receptor dimer is responsible for phosphorylation events (unlike PDGF RTK where they trans-autophosphorylate)
do all RTKs act as their own scaffold proteins
Yes, they all become phosphorylated on their tails, and they become that way to act as scaffolds
what are 3 proteins in PDGF receptor
PI 3 Kinase, GAP, phospholipase c-gamma (PLCy)
what do each of these signaling proteins do
play a role in PDGF receptor signaling
what do these signaling proteins recognize
phosphorylated tyrosines
what happens upon phosphorylation
creates binding sites that proteins have evolved to recognize and glue themselves on, formation of signaling complex
what do signaling proteins bind to specifically
not random phosphorylated tyrosines; signaling protein is recognizing a specific subset of phosphorylated tyrosines
what does PLC gamma recognize
2 phosphorylated tyrs at the tip: position 1009, 1021
what does GAP recognize
phosphorylated tyrosine at position 771
describe binding sites
not just any phosphorylated tyrosines; specific ones
so what specific piece of info do they look for when choosing which tyrosine to bind
AAs; tyrosines have neighbors and structural info along proteins
can serine and threonine be phosphorylated
yes; act as docking sites for proteins when this happens
in example what binds to phosphorylated tyrosine
SH2 domain
if a protein has SH2 domain what can it do
bind phosphorylated tyrosine
but SH2 domain is not just recognizing tyrosine; what else?
second binding site within Sh2 domain specifically recognizing AA very close to the one getting pohspohrylated
basically what happens w/ SH2 domains
recognizes phosphorylated tyrosine plus the amino acid right beside it
what do you get because of the large numbers of AAs
combinatorial diversity
describe the binding sites in Sh2 domain
2 binding pockets held closely together within binding site of protein; one for phosphotyrosine in question, other for amino acid side chain immediately next to it
what is the example pathway for RTKs
ras map kinase signaling pathway
G proteins are helpful for GPCRs, but what else
RTKs
what g proteins involved in RTKs
g alpha, beta, gamma
what g protein has GTP binding ability
g alpha
describe G alpha
large g protein
what is ras
small g protein; Ras GTPase
what is ras; in depth
ras monomeric GTPases (monomers, different from heterotrimeric G proteins)
what are examples of Ras superfamily
Rac and Rho
what does hyperactivation of Rho cause
forces cell to be contractile
what do Rac and Rho for ce cell to do
forces cell to make enormous lamellipodia (actin based structures for cell migration)
what are gtpases hella important in
signal transduction
what is one of main drivers of viral cancer and tumor growth
Ras
worst oncogenes
ras oncogenes; 30% of cancer has mutated form of ras
describe mutation in Ras
mutation in enzymatic site of GTPase
what does GTPase do
hydrolyzes GTP into GDP to turn it off
what happens if GTPase is mutated
can’t turn it of; always ON; keeps proliferating
what does GTPase Ras do
mediates signaling by most RTKs
in pdgf receptor what is stage 1
trans autophosphorylation of receptors (single site)
what is stage 2 in pdgf
multiple tyrosine sites phosphorylated on both receptors
(this is immediately after trans auto phosphorylation has occurred)
what next key player immediately after stage 1 and 2
Next key player: Grb2 (scaffold protein)
what does grb2 have
Has an SH2 domain, able to bind that now-phosphorylated tyrosine on the tail of that receptor
describe the SH2 binding
Not just recognizing that phosphorylated tyrosine, it’s recognizing phosphorylated tyrosine plus the amino acid immediately adjacent to it( that’s why its binding at the tip of the tail)
what is sh2 binding of tyrosine to tail of receptor dependent on
Activation dependent binding event
what type of protein is Grb2
adaptor protein (no enzymatic activity, but 2 Sh3 domains)
describe GRB2 structure
scaffold protein w/ single SH2 domain and 2 SH3 domains
what does Sh2 bind to
phosphorylated tyrosine in adjacent residue
what does sh3 bind to
SH3 domains are binding to proline rich regions on other signaling domains (blue rectangle)
what is indicator of Sh3 domain binding
AA sequence of signaling protein, and you see 3 or 4 or 5 prolines adjacent to each other but nothing in between
what is next thing that joins party
sos
describe sos
proline rich domains, has other binding sites
what is activity of sos
acts as a gef
what happens to sos upon binding
becomes active, now able to act as a Gef for inactive Ras
describe Ras protein
small GTPase protein, monomer (lipid anchor)
what is purpose of lipid anchor
to hold it against plasma membrane, right place and right time
what is inactive Ras protein bound to
GDP
what happens after GEF activity
conformational change denoted by outward bulge of protein, and red lines radiating away from it
what does inactive Ras bump into
interacts w/ Sos GEF domain that release GDP into cytoplasm (where it’s recycled back into GTP at some point)
what happens after GEF & conformational change
activates downstream signaling partners
what does Ras activate
MAP kinases
what does MAP stand for
mitogen activated kinase
where is active Ras protein
still anchored in plasma membrane
what does active Ras bump into
MAP kinase kinase kinase (Raf)
what does Ras bumping into raf do
triggers conformational change, raf is activated
are these kinases RTKs?
no; MAP kinase phosphorylates serine and threonine residues
what happens when Raf is activated
phosphorylates downstream targets
Raf phosphorylates twice to get
Mek
what comes after Mek
Erk
where is all this happening
plasma membrane (raf stuck at plasma membrane where ras is anchored)
what happens after Raf
all the steps after raf can go to cytoplasm
where is Mek floating around
Mek is floating around cytoplasm, bumps into active Raf and becomes phosphorylated
what happens after Mek is phosphorylated to Erk
Now they leave plasma membrane and go deeper in the cell
what does Erk act on
acts on diff targets to change protein activity and gene expression
what is ras map kinase pathway associated w/
cell proliferation
how many phosphorylation events per activation
2
how many receptors activated simultaneously in PDGF
multiple
how do you avoid jumbled signals or cross talk
scaffold proteins; diff pathways, diff downstream partners
both pathways in yeast share kinase A, what prevents them from getting singals wrong
scaffold 1 (mating) has binding site for Kinase A, then B, then C
scaffold 2 has kinase A, then itself has a kinase domain that activates kinase D
what does scaffold itself bringing substrate next to kinase A
in glycerol synthesis pathway it has no opportunity to phosphorylate anything else except next step in the pathway
what do Rho family GTPases do
functionally couple cell surface receptors to cytoskeleton
what is the output of signaling pathway controlling retraction
actomyosin contractility
what is actomyosin contractility
myosin-mediated actin filament contraction
what does actomyosin contractility cause
growth cone collapse (tries to have 2nd shot at getting where it wants to go)
what mediates growth cone collapse
contact depending signaling
where is ligand
anchored on surface of adjacent cell; already dimerized –> trans autophosphorylation (RTKs)
what is receptor
EphA4
what is ligand
ephrin A1
what does dimerized ligand do
dimerizes EphA4 receptor, trans-autophosphorylation of cytoplasmic tails
what is job of signaling proteins
phosphorylate Rho GEF
what is Rho
small GTPase, controls actomyosin contractility
what does phosphorylating Rho do
activate Rho by triggering exchange of GDP with GTP
what does activated Rho do
binds downstream effectors leads to massive actomyosin contractility and retraction of growth cone
where is ephexin
not bound to plasma membrane, anchored close enoughwh
where does rhoA diffuse
thru plasma membrane, gets contact w/ ephexin
what would happen if ephexin was mutated
no longer bound to receptor but it was still active, much less likely to be able to activate RhoA b/c its not in the right place
