cell signalling Flashcards
signal transduction
conversion of information from one physical/chemical form to another
(to pass on signal from signal molec to receiving cell)
agonist
any ligand/signal that activates a receptor
antagonist
ligand that blocks actions of agonist
–> competitive binding to receptor
desensitization
INactivation of receptor
(prepare for reception of new signal)
go to “clean slate”
cross-talk
interaction between signaling pathways
intermediates and products of pathways can affect behavior of other pathways
1st major illumination of (existence of) signalling pathways
studying retroviruses
– found that Rous sarcoma virus transmission could cause tumors (–> discovered basic signalling molecs bc studied the viruses)
proto-oncogene
normal cellular gene, NOT (yet) oncogenic.
= responsible for some part of cell proliferation/growth
(can become oncogenic if mutated)
oncoprotein
the actual protein (encoded by an oncogene) responsible for cancerous cell growth and proliferation
specificity
signaling molec must fit (and be specific/unique) to the binding site on its complimentary receptor
major features of signal transduction systems (6)
- specificity (high)
- affinity (high)
- sensitivity (high)
- amplification
- integration(must exist)
- desensitization/adaptation (must exist)
specificity determined by…
- complementarity between signal and receptor
- tissue/cell-specific receptor distribution
- tissue/cell-specific intracellular response system (vary in distribution)
Integration characteristic(s)
applies uniform response throughout environment
signal types
- hydrophilic (proteins, polypeptides, AA derivatives – epinephrine and norepinephrine)
- hydrophobic (cholesterol and fatty acid derivatives)
- both (dissolved gases – NO, CO)
characteristics of hydrophilic signal molecs
- can be stored
- short half-life
- receptors on plasma membrane
- indirect mechanisms
paracrine system
LOCAL signaling (w/in tissue/body region),
releases signal molecs to the extracellular space.
- lower affinity at receptors
ie: immune (cytokines), development
autocrine system
specialized paracrine signaling,
release local mediators, bind to same (own) cell type.
receptor type and proximity => specificity
7 steps in signalling
- synthesize signal
- release signal
- transport to target cell
- bind to receptor
- cell response
- remove signal (from receptor)
- terminate response
orphan receptors (type II)
non-steroid nuclear receptors, originally did not have known ligands! (now many do)
Selective Receptor Modulators (SRMs)
type of drug, = lab-engineered NR ligands,
are tissue-selective receptor agonist or antagonists.
*can also be MIXED (agonist and antagonist for different tissues at once)
ie: tamoxifen or raloxifene (for breast cancer)
mechanism of differential tissue-selectivity for SRMs
the NR changes conformation depending on which SRM binds,
–> diff. co-regulatory proteins bind to the NR
- ligand differences depend on small changes to sequence in “P-Box”
P-Box
the site at which the sequences of different SRMs vary most.
change p-box ==> change tissue-binding characteristics
DNA-binding mechanism for NRs
NRs bind to HREs (hormone response elements) on DNA
via Zinc fingers,
(type I: palindromes; type 2: direct repeats)
*few DNA seqs bind NRs, BUT most that DO encode transcription factors, so can influence more genes!
Hormone Response Element (HRE)
a sequence of DNA which binds NRs,
- palindrome (inverted repeat) for type I NRs
- direct repeat for type II NRs
*NOT unique across tissues/genes, get specificity elsewhere
PPAR alpha
NR in liver, binds fibrates;
regulates FA oxidation, lowers serum lipid levels;
(increase lipid oxidation, lower circulating triglycerides)
- dyslipidemia
PPAR delta (aka: delta-beta)
NR in adipocytes and other tissues,
has NO specific endogenous ligands;
increases FA oxidation, VLDL uptake, and Energy Expenditure;
- Dyslipidemia and obesity
PPAR Gamma
NR mostly in adipose tissue (also mm, macrophages)
binds glitazones (also FAs and eicosanoids);
increases adipogenesis (NEEDed for making white fat!),
lowers blood glucose levels, increases insulin sensitivity!
- preDM, obesity, metabolic syndrome
LXR
NR in liver, binds oxysterols; promotes secretion of cholesterol by - inhibit SREBP - increase bile acid synth by promoting gene for Cyp7a enzyme *atherosclerosis, dyslipidemia
FXR
NR in liver and intestine, binds bile acids;
promotes bile acid re-absorption (from ileum) by…
- increase # bile acid transporters in ileum
- inhibit gene for Cyp7a enzyme (bile synthesizer)
*high cholesterol and dyslipidemia
RXR (alpha, beta and gamma)
NR present throughout body,
binds 9-cis-retinoic acid.
*requires heterodimeric partner
– f(x) and associated diseases depend on which heterodimeric partner *accutane targets this NR!
Mechanism for NR type I
- hormone (ligand) crosses plasma membrane
- binds to NR/HSP complex, freeing HSP (chaperone)
- NR/ligand enters nucleus, binds DNA
- recruit co-activator
Mechanism for type II NRs
- hormone (ligand) enters nucleus (through nuclear pore)
- binds to NR on DNA
(NR = RXR-heterodimer) - NR complex swaps out co-repressor for co-activator
NO (nitric oxide) synthesis
substrate: argenine
product: citrulline and NO
3 NO synthases:
- Neuronal Constitutive NOS (nNOS)
- Endothelial constitutive NOS (eNOS): in smooth mm.
- Induced NOS (iNOS): in macrophages
NO as signal molec
gas, free radical, VERY short half life,
does NOT bind classically.
regulates:
-relaxation of smooth muscle (vascular) tone
-immunological f(x) (antimicrobial)
destructive characteristics of NO
free radical,
binds to any cell structures and interrupts cellular processes
–> kills the cell and neighbor cells
intracrine signalling
signalling both withIN and between cells,
only example: NO
medical use of nitroglycerin
used to treat angina
- releases 3 NOs
endothelial releasing factor
= Nitric Oxide
4 types of binding domains (for modular proteins)
PH = pleckstrin homology domain PTB = phosphotyrosine-binding domain SH2 = Src homology 2 domain SH3 = Src homology 3 domain
2 examples of GPCR effectors (G-protein coupled receptors)
- adenylyl cyclase
2. phosphoinositide cascade
structure of a GPCR
7 transmembrane domains,
- effector molec (IN membrane)
- g-protein (cytoplasmic side of membrane) –> “trimeric complex” (alpha, beta, gamma), *alpha separates from beta/gamma to f(x)
- receptor w/ NH2 tail (on extracellular side of membrane)
response of receptor depends on…
strength, duration and timing of signal
hydrophobic signaling molecs
proteins, polypeptides, AA derivatives (NE/Epinephrine)
stored in secreting cells, long half life,
direct mech, intracellular Rs
mostly type II NRs
hydrophilic signaling molecs
cholesterol and fatty acid derivatives, (steroids)
NOT stored, short half life,
INdirect mech, plasma membrane Rs
mostly type I NRs
“Both” hydrophilic and hydrophobic signal molecs
gases NO and CO2.
covalently bind to the receptor (guanylyl cyclase R)
7 steps of cell signaling
- Synthesize ligand, 2. Release signal (ligand)
- Transport to receptor, 4. Bind to Receptor
- Response
- Remove ligand, 7. Terminate response
when cell does not receive the appropriate signals
induces apoptosis
(cell suicide)
bc can’t continue growth, cell cycle, etc. w/o signals!
types of pathway desensitization
- Receptor sequestration
- Down-Regulation of Receptor (internalize and degrade R)
- receptor INactivation
- INactivate 2nd messengers (downstream)
- make INhibitory protein for 2nd messengers
3 parts to a cell receptor
(= protein)
- extracellular domain
- transmembrane domain
- intracellular domain
* each receptor has >1 functional domain!
type I NR characteristics
(steroid TFs – ligands = steroid derivs),
form homodimers, bind to palindromes.
LOW concentration in blood
(–> high affinity, R has high specificity)
Type I NR major ligands
= steroids (cholesterol derivatives).
- progesterone
- estrogen, estradiol; tamoxifen/raloxifene
- androgen (testosterone)/DHEA
- aldosterone (mineralcorticoids)
- cortisol (glucocorticoid)
Type II NR major ligands
(proteins and polypeptides)
- PPAR alpha and gamma
- Xeonbiotic receptor (PXR, CAR-beta)
- bile acid receptor (FXR)
- oxysterol receptor (LXR)
NR type II characteristics
(“dimeric orphan receptors”)
form heterodimers w/ RXR,
bind direct repeats.
high concentration in blood (“promiscuous”)
–> low receptor specificity and affinity
AF1 domain of nuclear receptor
activator factor 1,
activated by signal in ABnormal state
(ie: cancer cells)
* at N end of protein
AF-2 domain in nuclear receptors
activator function 2,
activated by ligand binding in normal/healthy cells.
*near Carboxyl end of the protein, part of ligand binding domain
DNA binding Domain (“DBD”) of nuclear receptors
in middle of receptor protein, binds DNA (HRE site if type I) when R = activated.
best target in NR pathway for cancer treatment
Co-regulators!
- inhibit co-activators
or stimulate co-repressors.
–> help w/ tissue-targeting, minimizing side effects
Ligand Binding Domain (LBD)
site on NR where ligand binds to activate the protein.
- nearer C end,
contains AF-2 domain
Examples of Cytokine Receptors (ligands)
- Growth hormone
- erythropoeitin
- prolactin
- Leptin
- Interferon (alpha/beta/gamma)
Cytokine Receptors (tyrosine kinase receptors)
NOT enzymes, = receptors for cytokine ligands
families: Src, JAK, STAT
mTOR
serine/threonine kinase,
promotes cell growth and protein synthesis.
activated by AKT
receptor tyrosine kinase (RTK) mechanism
- INactive: in membrane as monomer
- ligand binds, Rs dimerize (except Insulin R = already dimerized)
- activate intracellular tyr. kinase domain
3.tyrosine kinases cross-P (auto-P) the opposite receptor - increase kinase f(x)/make docking station for signal molecs.
- more phosphorylation–> activate downstream pathways
(Src, PI, PI3-K, MapK)
Receptor Tyrosine Kinases (RTKs)
cell signal receptors,
set of 2 Rs on 2 intracellular proteins w/ tyrosine kinases.
Activate when ligands bind –> auto-phosphorylate and dimerize.
ligands: EGF, FGF, insulin (growth factors!)
pathways activated: Src, Phospholipase C, MAP kinase, PI-3K
Phospholipase C cascade
initiated by RTK, activates Phosphoinositide (PI) pathway (w/ GPCRs) --> increase cytosolic Ca2+ levels (boost signaling w/ Ca2+) ** directly makes DAG and Ins-1,4,5-P3
Src pathway
= cytosolic tyrosine kinase enzyme,
starts of signal cascade to increase cell motility, decrease adhesion (regulate cell f(x)).
activated by RTKs and Cytokine Receptors
Ras proteins
a GTPase signal molec;
activated by RTKs (= freed from plasma membrane),
relay cell proliferation signals to MAP kinase cascade.
* + reg: GEFs
- reg: GAPs
“GAP” protein (GTPase activating protein)
INactivate Ras signal molec.
(increases rate of hydrolysis of GTP on Ras)
* Binds directly to the RTK,
(helps return Ras to inactive when RTK is inactive).
“GEF” (guanine exchange factor)
increase Ras function;
–> promotes exchange of GDP and uptake of GTP to Ras
(when active, increase rate of f(x))
MAP kinase cascade (mitogen activated kinase)
- a serine/threonine kinase cascade, longer lived than RTKs*
1. Ras activates Raf-1 (MAP kinase kinase kinase)
2. Raf-1 activates Mek (MAP kinase kinase)
3. activate MAPK * by dual +P of tyrosine and threonine - -> activate more… –> promote cell proliferation
- ** activates Myc, Jun, Fos Tfs!***
Insulin Receptor Substrate (“IRS”)
Insulin-specific receptor that binds to RTK (when RTK = activated),
=> docking station for mTOR, AkT, and MAPK
mitogens
cell signaling moles,
induce cell proliferation
ie: Growth Factors, estrogen
cytokine receptors (tyrosine kinase-coupled receptors)
ligands: growth factors, prolactin, erythropoietin, cytokines (interferon-gamma)
R is NOT enxyme, relies on tyrosine kinases from cytoplasm to phosphorylate the R and start cascade (Srcs/JAKs)
Activate: STATs (transcription factors)
Cytokine
a signaling molec that binds to tyrosine kinase-coupled receptors (aka cytokine Rs)
- promotes hematopoeitic differentiation and protection against viral infection (interferons)
Cytokine receptor mechanism
- cytokine binds to 2 Rs –> dimerize
- R-bound JAKs (= tyr. kinases) cross-phosphorylate the each other
- JAKs phosphorylate the R
- STATs bind to R
- STATs = phosphorylated by JAKs
- STATs dimerize, release, go to nucleus to upregulate gene transcription (hematopoiesis, immune function–> Myc/Fos)
STAT
family of signal proteins activated by cytokine R/JAK complex.
dimerize when tyrosines = phosphorylated,
go to nucleus to upregulated gene transcription of
- hematopoiesis - mammary gland dvpt
- immune F(x) Myc/Fos
pathways activated by cytokine receptors
- STAT and same as RTKs... - INS - MapK - Src - Phospholipase C (gamma) - PI-3K
Epo
signal molec, activates EpoR (a cytokine R)
promotes red blood cell formation
* often used for blood doping*
SHP phosphatase
protein that INactivates JAKs
if def., will have extra EpoR activity –> extra high RBCs
ANPs (Atrial Natiuretic Peptides)
signal hormones released by heart, bind to guanylyl cyclase Rs (NPRs). promote smooth muscle relaxation in blood vessels --> vasodilation. *also: BNP (brain), CNP (cardiac)
receptor guanylyl cyclase (aka NPR – natiuretic protein R)
Receptor for ANPs, (“A” for ANP/BNP; “B” for CNP)
has cytoplasmic guanylyl cyclase enzyme to make cGMP.
cGMP activates PKG (protein kinase G)
–> cascade, ie: to inactivate IP3 –> sm. muscle relaxation
Maroteax dysplasia
a type of short-limbed dwarfism caused by mutations in NPR-B
Natiuretic Protein Receptor B
TGF-beta receptor lignds
TGFbetas, BMPs (bone morphogenetic proteins), anti-mullerian hormones, inhibins, activins.
paracrine signaling (held in ECM until released locally)
complex, may homo- OR hetero-dimerize
BMPs
Bone morphogenic proteins,
ligand for TGF-beta Rs
TGF-Beta Receptor
aka: serine/threonine kinase receptor
w/ activation by ligands, phosphorylate SMADs,
SMADs go to nucleus to alter gene transcription.
Regulate: cell prolif/death, immune response, homeostasis
** inhibits cell prolif., = tumor suppressor! ***
TGF-beta R mechanism
- ligand binds to TGF-Beta RII, activates
- Receptor II activates and dimerize w/ receptor I
- serine/threonine kinase of RI phosphorylates SMAD
- activated SMAD goes to nucleus as TF
(inhibit cell prolif, prevent tumors)
Canonical Wnt Pathway
signaling pathway in cell to regulate cell adhesion;
* regulates beta-catenin proteolysis (ubiquitination).
Also activates: Phospholipase C and Dsh pathways.
ligand: Wnt; Receptors: Frizzled and LRP
*INactive –> beta-catenin degradation.
INactive (basal state) Wnt Pathway
Receptors not dimerized, no “disheveled” released.
- -> beta-catenin = sequestered in cytosolic degradation complex, where phosphorylated (marked for Ubiquitination and degradation).
- —> Groucho corepressor inhibits Wnt target genes (ie: Myc)**
Beta-catenin Degradation Complex
in cytosol, contains 4 proteins: - GSK3, CK1 = ser/thr kinases (+P to B-cat. to mark for ubiquitination and degradation) - axin, APC = scaffold proteins
Groucho
corepressor for Wnt genes,
binds to LEF1/TCF when Wnt pathway = inactive
to inhibit Wnt target genes (ie: myc)
Mech of Wnt pathway activation (and f(x))
- Wnt binds to Frizzled R –> cluster Frizzled and LRP Rs
- activate (phosphorylate) Disheveled protein
- recruit degradation complex to receptors
—> Wnt free to go to nucleus to promote target genes (ie: Myc)
(promote cell proliferation, etc)
“APC” (Adenomatous Polyposis Coli)
familial colon cancer from mutation in APC,
causes formation of MANY polyps in colon, appears in early adult.
–> no Wnt degradation, so excessive cell proliferation!
PTEN
regulatory lipid phosphatase, dephosphorylates PIP3.
–> blocks enzymatic activity (no AKT activation)
= tumor suppressor
PI-3K (phosphoInositol-3-kinase)
Enzyme activated by RTKs,
PIP2 –(add P at 3 position)–> PIP3
–> PIP3 recruits AKT for phosphorylation by PDK1
** PIP3 –(PTEN)–> PIP2
AKT
signal enzyme downstream of RTK,
recruited to membrane by PIP3, activated (+P) by PDK1.
** +P to many more downstream proteins (starts cascade)
(aka: protein kinase B)
PDK1
enzyme, part of RTK/PIP3 signal pathway/complex. activates AKT (+P) after recruited by PIP3
downstream molecs phosphorylated by AKT
activated: MDM2, mTOR, NF-kB
INactivated: BAD
mTOR
ser/thr kinase complex, activated by phosphorylation from AKT.
promotes cell growth, glycolysis/glucose transport, and protein synthesis (= oncoprotein)
inhibits p53
* stimulated by growth factors, inhibited by Rapamycin
BAD
regulatory enzyme inhibited by AKT,
active: sequesters Bcl2 –> so promote apoptosis
(Bcl2 blocks apoptosis)
if: active AKT –> INactive BAD –> active bc12 –> NO apoptosis
function of g-protein alpha subunit
= GTPase,
hydrolizes G-protein to turn signal off
(desensitizes)
G-protein in GPCRs (f(x))
transduces signal from receptor to effector.
w/ GTP bound: ON
w/ GDP bound: OFF
*alpha subunit INactivates the signal (swaps GDP for GTP)
isoprenoid unit
cholesterol derivative,
anchors G-protein to cytosolic side of plasma membrane
characteristics of second messengers
- short half life (seconds)
2. high affinity for target
GRK (g-protein receptor kinase)
desensitizes GPCR,
phosphorylates receptor so Arrestin (inhib. molec) will bind.
aka: beta-ARK (beta adrenergic receptor kinase)
cAMP phosphodiesterase
converts cAMP to 5’-AMP,
= inactivates cAMP
(cAMP made/activated by adenylyl cyclase)
AC (adenylyl cyclase) pathway
AC –(activate)–> cAMP,
2 cAMP –(activate)–> PKA –> (phosphorylate target proteins or cAMP Response Element “CREBP”)
CREBP
cAMP Response Element Binding Protein,
a transcription factor
binds CBP (CREB binding protein), which is activated by PKA
(part of GalphaS pathway, w/ Adenylyl cyclase)
mech. of pertussis toxin
binds and blocks Galpha-i
–> overactive AC –> too much PKA
mech. of cholera toxin
blocks f(x) of g-protein alpha subunit
- -> no deactivation of Gsalpha
- -> overactive AC –> too much PKA
protein kinase C
family of ser/thr kinases,
used in signaling cascades to phosphorylate target molecs.
(activated by DAG&Ca2+ from GPCRS, cGMP from NPRs)
promote cell proliferation
DAG (diacylglycerol) in signaling
- w/Ca2+, activated PKC
- precursor for eicosanoids and prostaglandins
(made from PIP2 in PI pathway)
PI (phosphoInositide pathway)
activated by GPCRs, **ligs: Ach, NE, Epinephrine, Ghrelin**
1. PLC-beta (phospholipase C) --> PIP2
2. PIP2 --> IP3 and DAG
3. IP3 --> activate Ca2+ channel
DAG (+Ca) --> PKCmajor cellular proteins that bind Ca2+
- PKC (phosphokinase C)
2. calmodulin (“CaM”)
enzymes that bind CaM (calmodulin)
- eNOS/nNOS
- PKC
- phosphorylase kinase (regulates glycogen phosphorylase)
major (relevant) GPCR ligands
- Ghrelin (activates PI and AC pathways at same time!)
- MSH-alpha (POMC neurotransmitter)
- GLP-1
- external signals (light/sound/pressure)
more: Ach, glucagon, CCK, LH, prostaglandins…
