Exam 4 Flashcards
what was the first second messenger discovered
cAMP
what are the types of signaling
juxtacrine (contact-dependent)
autocrine (act upon self)
paracrine (signal in immediate surroundings)
endocrine (signal in bloodstream, far away)
synaptic (NTs, neuron-neuron or neuron-cell)
what is an example of diff responses to the same ligand
Acetylcholine
in heart, acts on muscarinic receptor, slows HR
in salivary gland, acts on muscarinic receptor, causes secretion
on skeletal muscles, acts on a diff receptor, causes contraction
main diff between extra and intracellular ligands
extra - hydrophilic
intra - hydrophobic (and small)
explain amplification
each binding event can trigger multiple downstream molecules, allowing for exponential propagation of the signal
also allows for better regulation than a 1:1:1 reaction
relative speeds of signaling mechanisms:
channels, catalytic, G-protein, nuclear
channel: milliseconds
catalytic: seconds/mins
G-protein: seconds/mins
nuclear: hours (receptor triggers DNA transcription
where do steroid hormone receptors bind?
the LBD region of the receptor, inhibiting the inhibitor, then the signal is translocated to the response element on the DNA inside the nucleus
what does the coactivator do in the steroid hormone receptor?
binds to the ligand receptor, allowing for conformational changes that allow for transcription of the target gene
explain the diffusible messenger molecule of NO
when acetylcholine is taken up by the GPCR on endothelial cells, it is converted to Phospholipase C –> IP3 –> Ca/calmodulin –> NO synthase –> NO is released onto muscle cells leading to relaxation of the muscle cell
what type of structure do G proteins have
heterotrimeric (G-alpha, -beta, and -gamma)
on in the presence of GTP off in GDP
7 transmembrane regions
protein kinase vs phosphatase
kinase: phosphorylates protein
phosphatase: dephosphorylates protein
what are the ways in which a signaling complex is formed?
preformed (already held together by scaffolding before, activated in presence of ligand)
assembly (recruited after ligand binds receptor)
what are phosphoinositides
bound to membrane, can mind intracellular proteins that will hyperphosphorylate them and lead to downstream effects
what does GEF do
promotes dissociation of GDP, allowing for GDP/GTP exchange (does not phosphorylate, just causes GDP to dissociate and there is a higher conc of GTP in cell)
what is an example of a GEF
Sos
where does Sos/GEF bind?
SH3 domains on GRB2
where does GRB2 bind the EGF dimer
SH2 domain
agonist vs antagonist
agonist binds receptor, eliciting a similar response to ligand bind receptor
antagonist binds receptor in same or diff spot, inhibiting receptor
why was it so hard to study signal transduction
there are so many molecules it was impossible to know which were interacting. needed better purification methods
which subunit of GPCRs bind the GTP
alpha
what are the effects of GPCRs?
enzymes: adenylate cyclase, phospholipase C (PLC), phosphodiesterase (PDE)
ion channels: potassium (GIRK) and calcium (VDCC)
what are the regulators of GPCR signaling
aka RGS (regulators of GPCR signaling)
GAPs (GTPase activating proteins)
what do GAPs do
enhance inactivation
promote signals
what do GRKs do
GPCR kinases
phosphorylate agonist bound receptor
have seven genes
what do arrestins do
uncouple receptors form G-preteins, “arrest” signal
act as scaffolding, link GPCRs to other pathways (MAPK, NFkB)
what explains the different families of GPCRs and what features do they have
convergent evolution
7 TMs, conserved regions/domains
include the Rhodopsin, secretin/adhesion, and metabotropic families
key features of the 7TM GPCRs
N terminus = outside
C terminus = inside
N-linked glycosylation = sugar modification
Cys residues outside = disulfide bonds, stabilize TM domains
Cys residues inside = palmitoylation, anchor C-term to membrane
Ser/Thr = inside, kinase
phosphorylation sites
where are the most highly conserved areas of a GPCR
the 7TM regions
how can GPCRs be activated
protease/cleavage
glycoprotein hormones
neurotransmitters
all maintain the same basic shape and 7TM regions
what is rhodopsin
GPCR
associated with light intake in the retinas
7TMs form a pocket though which photons can pass through
opens pocket when activated
agonist and antagonists effects
neutral antagonists keep signal at basal level
inverse agonists dec basal level
partial agonists inc receptor response, but less than natural ligand
full agonist completely mimics response as if it were a natural ligand
G protein cycle
1) resting G-alpha/GDP, beta, and gamma all bound together
2) ligand binds receptor
3) receptor activates and GDP dissociation from alpha
4) GTP binds alpha
5) G-alpha/GTP dissociates from beta/gamma
6) GTP hydrolysis (returns alpha/GTP back to alpha/GDP, which reassociates with beta/gamma)
which steps are irreversible in the g protein cycle
alpha/GTP dissociation from beta/gamma
pushes cycle forward
types of G protein subunits
alpha, beta, and gamma
there are many types of ea (many alpha, many beta, many gamma), but you need at least one of each to make a full g protein
not all types work with ea other
key features of G-alpha
GTP binding domain
N terminus palmitoylation or myristoylation (binds membrane)
key features of G-beta
unstable without gamma, forms very tight bond
key features of G-gamma
stabilizes beta
C terminus isoprenylation (binds membrane)
which G protein subunit binds receptor
alpha
what do most isoforms of adenylate cyclases have
TM domains (9/10 isoforms have them)
structure of adenylate cyclase
12 TM domains (2 repeats, ea with 6 parts)
C1 and C2 in cytoplasm allows for catalytic activity
what is the classical GPCR pathway
activated GPCR > activated G-alpha activates adenylyl cyclase > adenylyl cyclase forms cyclic AMP > cAMP activates PKA > PKA is recruited into the nucleus > PKA activates CREB > CREB binds CBP (CREB binding protein) > CREB/CBP binds the CRE (cAMP response element) on DNA > transcription
what are the mechanisms downstream of cAMP
PKA (classical)
cyclic nucleotide-gated ion channels
Epac (exchange proteins activated by cAMP)
what are the second messengers of Phospholipase C (PLC)
DAG and IP3
PLC cleaves PIP2 into IP3 and DAG
what does DAG do
activates PKC
what does IP3 do
release Ca from the ER
GPCR PLC pathway
GPCR activated > G-alpha gains GTP > G-alpha activates PLC > PLC cleaves PIP2 into IP3 and DAG > IP3 opens ion channel on ER, releasing Ca > DAG binds PKC, PKC binds Ca > PKC is activated
what domains do RGS proteins have
RGS box
localization and protein interaction domains
what do RGS proteins do
dephosphorylate GTP back to GDP (pushes pathway forward)
when bound to RhoGEF, promotes exchange of RhoGDP for RhoGTP on RhoGEF (downstream cytoskeleton effects), still the G-alpha is having GTP turned to GDP
what associates with arrestins
GRKs
what do GRKs do
after ligand has bound GPCR, facilitates binding of arrestin (stops response)
arrestins can also act as scaffold for other proteins that can lead to alternative pathways
what is desensitization
GRK-arrestin mediated inhibition of GPCR signaling
causes endocytosis of GPCR
what is the GRK pathway
GRK phosphorylates ligand bound receptor > recruits arrestin > arrestin binds, recruits AP2, ERK, and clathrin > receptor is endocytosed
what molecules does arrestin recruit
AP2, ERK, clathrin
what are the downstream effects of arrestin bound GPCRs
desensitization
internalization
scaffold for other pathways
Ubiquitination
what are the implications of Arr1 and Arr2
more regulation, some have opposing effects in diff arrestin pathways
what do biased agonists do
promote conformational changes in receptors that prefer activation of specific signaling pathways (one ligand favors a specific pathway)
what are the pharma implications fo biased agonists
they are more specific and favor one GPCR pathway, limit off target effects
themes of tyrosine kinase signaling
conversion of an extracellular signal to changes in intracellular tyrosine phosphorylation.
movement of signaling molecules to the membrane.
induces protein-protein interactions through specialized protein motifs.
what is a non receptor TK
a TK without TM regions
features of Src
first discovered in viruses (v-Src), human (c-Src) has high homology, first TK discovered
nonreceptor TK
key domains of Src
kinase (SH1), SH2, SH3, unique domain (SH4)
2 phosphorylation sites (both tyrosines)
myristolation, palmitolation, autophosphorylation
what is the diff between the two phosphorylation sites on Src
Tyr 416 (on kinase/SH1/activation loop) turns on Src
Tyr 527 (on C terminal tail) turns off Src
how does Src become membrane associated
originally hydrophobic, post translational modifications:
cleavage of methionine (1st aa, now starts with glycine), myrisolation of that glycine, can have palmiltoyltion
describe inactive Src
SH3 binds proline rich region
SH2 binds inhibitory P on C terminus, activation loop is blocked
phosphorylation of inactive site, dephosphorylation of active site
describe active Src
SH2 releases inhibitory P, SH2 binds to RTK
fully active when active when kinase Tyr is autophosphorylated
phosphorylation of active site, desphosphorylation of inactive site
what are the diff types of interactions in active vs inactive Src
active = intermolecular (SH2 binding RTK)
inactive = intramolecular (SH3 with proline rich region, SH2 with inhibitory P-Tyr)
what is Csk and how does it work
tumor suppressor gene that is able to phosphorylate the inhibitory P on Src, dec cell proliferation
key features of RTKs
TM proteins with ligand binding outside and kinase inside
dimerization and cross phosphorylation
numerous downstream pathways (transactivation)
regulate their own inactivation through Cbl
general steps in RTK activation
1) Ligand-induced conformational change transmitted from the extracellular to intracellular domain
2) Tyrosine phosphorylation (cross-phosphorylation) of C-terminal tyrosine residues (or phosphorylation of a scaffold; e.g. IRS1, GAB14,..)
3) “Relaxation” of the “activation loop” to allow access to the active site
4) Further tyrosine phosphorylation of the C-terminal domain to provide docking sites for SH2 and PTB domain-containing proteins
what are the common domains in Src, PLC, and Grb2
SH2 and SH3
what is the main difference in PTB domain and SH2 domain binding
SH2 ligand binds in pocket
PTB ligand augments beta sheet
what do SH2 domains bind
tyrosine-phosphorylated sequences
what do SH3 domains bind
proline rich regions
what does the PH domain bind
PIP3
what is IRS and what does it bind
insulin receptor subunit
acts as an adapter protein to mediate signaling
PH domain binds PTB, which binds PIP3 and other SH2 domain proteins
SH2 domain proteins
PIP3, Grb2
what does Cbl do
E3 Ub ligase
after EGFR is autophosphorylated, Tyr phosphorylation triggers Ub, gets degraded after being internalized
PI3K/Akt pathway overview
RTK phosphorylates PI3K > PI3K phosphorulates PIP2, converting it to PIP3 > PIP3 binds PH domains, can act on both PDK1 and Akt/PKB > PIP3 recruits both Akt and PDK1 allowing them to interact > PDK1 phosphorylates Akt > Akt phosphorylates other downstream proteins > affect survival, proliferation, and motility
what is an inosital ring
head group that is inserted into the membrane
ex: PIP2, PIP3
what converts PIP2 to PIP3
PI3K
what converts PIP3 to PIP2
PTEN
what are the important domains in p110
Adapter binding, Ras binding, C2, Helical, Kinase
what are the importnat domains in p85
SH3, Rho-GAP, nSH2, cSH2
what does p85 bind
PI3K at the PH domain
if it is insulin, p85 binds IRS instead of directly to PI3K
also binds p110, which in turn can bind Ras
where does PIP bind PLC
PH domain
