what is signal transduction
process by which cells receive & respond to cues from biological environment
essentialy for normal development, homeostatis
if wrong/aberrations, get disease like: cancer, ID, developmental genetic aberration, psychiatric disease, diabetes
target for most drugs = signal transduction
general categories of cell-cell signaling? how do they differ?
1) endocrine: cell produces hormone, it enters into circulation, travels long distance > target cell; i.e. pituitary-synthesized hormone
2) paracrine: signaling cell releases factor into local area; ddoesn't enter bloodstream; i.e. SHH
3) neuronal: specialized paracrine signaling w/ neutrotransmitters
4) contact-dependent: action limited to cell adjacent, cells must be in contact to hit target cell
5) autocrine: cell both makes signaling factor & responds to it; i.e. cancer cells
types of signaling factors?
amino acids/their derivatives
drugs of abuse
what is the basic principle of cell-surface receptors?
high affinity btwn hydrophilic signal molecule & cell-surface receptor makes them bind
causes signal trasnduction event w/in cell; just diffusion wouldn't work
describe process of FAST signaling
what does it cause?
extracellular signal molecule > cell-surface receptor protein > intracellular signaling pathway > altered protein function > altered cytoplasmic machinery > altered cell behavior
< sec to mins
occurs in CYTOPLASM only
describe process of SLOW signaling
what does it cause?
extracellular signal molecule > cell-surface receptor protein > nucleus > altered protein synthesis by changing transcriptional program > altered cytoplasmic machinery > altered cell behavior
SLOW: mins to hrs
occurs in both CYTOPLASM and NUCLEUS
major classes of cell surface receptors?
1) G-protein coupled receptors (GPCRs)
2) Receptor tyrosine kinases (RTKs)
3) Cytokine receptors
what transmits through GPCRs (what are the LIGANDS)?
neurotransmitters (epinephrine, serotonin, dopamine)
sensory stimuli (light, odorants)
many prescription drugs
what is the structure of the GPCR?
how does it basically work?
protein w/ 7 transmembrane domains
ligand binds the receptor extracellularly, causing conformational change in the receptor
change allows intracellular domain to interact w/ heterotrimeric G protein
what is the structure of the heterotrimeric G protein?
what is each subunit able to do?
has 3 subunits: alpha, beta, gamma
beta-gamma are always found together
alpha is able to bind GT & to hydrolyze bound GTP to GDP
alpha and gamma have post-translational fatty acid residue connected - can insert into inner surface of plasma membrane
describe activation pathway of the G protein
1) heterotrimeric G protein is inactive when bound to GDP, all 3 subunits are interacting then
2) ligand binds GPCR > receptor changes conformation to allow interaction w/ alpha subunit of G protein
3) alpha subunit releases GDP, binds GTP
4) alpha subunit w/ GTP dissociates from beta-gamma subunit
5) alpha-GTP and beta-gamma subunits now interact w/ downstream receptors
what causes alpha subunit of G protein to switch GDP for GTP when activating?
[GTP] > [GDP] in cytoplasm
so, if GDP is expelled, GTP replaces it
describe the pathway for inactivation of the G protein
1) after activating its target protein, alpha subunit hydrolyzes GTP to GDP, with aid of an RGS, regulatory of G-protein signaling
2) GDP-bound alpha subunit is inactive
3) inactive alpha subunit re-associates w/ beta-gamma subunit b/c GDP-alpha has affinity for beta-gamma subunit
what is RGS? what is its function?
membrane-bound regulatory of G protein signaling protein
increases GTP-ase activity of alpha subunit of heterotrimeric G-protein, allows it to inactivate and "reset" much faster
aka helps alpha subunit hydrolyze GTP to GDP
how many GPCRs are there, and how many different alpha/beta/gamma subunits?
1000 GPCRs, 25 alpha/beta/gamma subunits
so many different GPCRs will work thru similar pathways to the subunits
what are small or secondary messengers?
a non-protein/enzyme intermediate molecule that acts as an intermediate to propagate a signal from a receptor
i.e. adenylyl cyclase, cAMP, IP3, DAG, Ca
what are the 3 classes of G-protein's alpha subunits, what do they directly do?
Gs: activates adenylyl cyclase
GI: inactivates adenylyl cyclase
Gq: activates phospholipase C
describe the pathway of GS on its downstream targets
1) activated G-alpha-S activates adenylyl cyclase
2) adenylyl cyclase converts ATP to cAMP
3) cAMP activates targets, like PKA
4) cAMP binding to PKA activates it by dissociating its regulatory subunits
5) PKA phosphorylates targets, i.e. glycogen phosphorylase & glycogen synthase
what is a kinase? what is a protein kinase?
kinase: enzyme that phosphorylates, usually from ATP to a substrate
protein kinase: enzyme in which substrate is a protein
what are the 2 major types of protein kinases?
1) kinases that phosphorylate Ser/Thr residues
2) kinases that phosthorylate Tyr residues
what is the structure of PKA?
how is it activated?
PKA has 4 subunits: 2 regulatory, 2 catalytic
when cAMP binds to the 2 regulatory subunits, they no longer have affinity for catalytic subunits
catalytic subunits go forward & phosphorylate substrates, propagate signal
describe pathway involving adrenaline and GS
what is the result? and what kind of response is this?
1) situation of fight/flight, adrenaline binds GPCR, activates GS
2) GS binds & activates adenylyl cyclase
3) adenylyl cyclase converts ATP to cAMP
4) cAMP activates PKA
5) PKA phosphorylates & activates phosphorylase kinase
6) phosphorylase kinase phosphorylates phosphorylate glycogen by hydrolyzing ATP to ADP
7) phosphorylated phosphorylate glycogen causes glycogen breakdown
this gives your muscles energy to run!
what steps in the GS/adrenaline pathway are sites of amplification? which are not sites of amplification?
Amplification sites (>1:1 ratio of activator>activated):
adenylyl cyclase activates cAMP
PKA phosphorylates substrates
Not amplification sites (1:1 ratio of activator>activated)
GS activates adenylyl cyclase
cAMP activates PKA
what is meant by a site of amplification?
signaling such that limited amount of information input causes explosive burst of information output
what is signal branching? example?
when 1 receptor protein can activate more than 1 kind of G protein
i.e. PKA has numerous substrates
why are signal transduction pathways reversible? what are examples of this?
because if signaling goes awry, that can result in disease
1) alpha subunit + RGS cause GTP hydrolysis to GDP
2) cAMP phosphodiesterase digests cAMP to AMP, which is a biologically inert signaling molecule
3) protein phosphotases inactivate substrates that require phosphate for activation
describe the phospholipase C / Gq pathway
1) signal molecule activates its GPCR
2) Gq subunit of G protein binds & activates phospholipase C-beta
3) phospholipase C breaks down the membrane phospholipid PIP2 into IP3 and DAG
4) IP3 can activate release of Ca2+ inside the cell, which activates PKC, which causes other signaling functions
DAG directly activates PKC also
what are IP3 and DAG examples of?
from the phospholipase C pathway
what is the function of Gi?
inhibits adenylyl cyclase
describe activation and pathway of Gt
1) rhodopsin receptor activates Gt
2) Gt activates cGMP phosphodiesterase
3) cGMP phosphodiesterase breaks down cGMP
describe pathway of cholera toxin
1) cholera toxin is an enzyme. it ADP-ribosylates GS-alpha
2) ADP-ribosylated GS cannot hydrolyze GTP to GDP now
3) GS w/ GTP thus remains permanently activated, keeps activating adenylyl cyclase
4) activated adenylyl cyclase increases cAMP levels, activates PKA
5) prolonged PKA activation in intestinal epithelium causes efflux of Cl- and water into gut
causes copious, watery diarrhea
describe pathway of pertussis toxin
1) pertussis toxin ADP-ribosylates alpha-subnit of Gi
2) prevents Gi from binding to GPCRs
this inhibits the inhibitor
3) leads to increased [cAMP]
causes bacteria colonization, leads to whooping cough
what are 3 ways in which the GPCR to PKA can be shut down?
1) Gi activation, as it inhibits adenylyl cyclase, which activates cAMP which activates PKA
2) Block Gs from activating adenylyl cyclase
3) Block cAMP, as that is what adenylyl cyclase activates, and cAMP then activates PKA
what is the generalized structure of an enzyme-coupled receptor?
- receptor with extracellular ligand-binding, membrane-spanning, and intracellular regions
- receptors are dimerized (brought together) by ligand binding
- receptors may be homodimers (same protein) or heterodimers (different proteins)
- enzyme activity may be intrinsic to receptor, or intracellular region may bind to receptor non-covalently
what is the largest family of enzyme-coupled receptors?
what is its structure?
receptor tyrosine kinases
extracellular: ligand-binding domain
single membrane spanning region
intracellular: tyrosine-kinase domain
ligands of receptor tyrosine kinases (RTKs)?
platelet-derived growth factor
epidermal growth factor
fibroblast growth factor
describe signal transuction for receptor tyrosine kinases (RTKs)
RTKs exist as very weakly inactive in plasma membrane
1) ligand binds RTK
2) dimerization causes receptors to be phosphorylated (auto or trans)
1st: phosphorylation of tyrosine-kinase domain
2nd: phosphorylation outside of tyrosine-kinase domain
receptors are now active
3) phosphates provide binding sites for next factors in signal cascade, and activates receptor's catalytic function
what are the classic domains of RTK signaling?
what does each bind?
SH2: binds phosphotyrosine regions
SH3: binds polyproline regions
specific factors' SH2 or SH3 domains bind to phosphate of specific Tyr residues
what are examples of modular enzymes?
what's their function?
modular enzymes: contain SH2 and SH3 domains + an enzymatic component
PLC-gamma: binds to RTKs
PLC-beta: binds GQ of G-protein alpha subunit
what are modular adaptor proteins? their function?
modular signaling protein with SH2 and SH3 domains but NO ENZYMATIC activity
what is Ras?
when is it active/inactive?
how does it switch from active to inactive state?
Ras: s small G-protein which can bind GTP and GDP, has GTPase activity
when GTP-bound: active; GDP-bound: inactive
when signal enters, GEF (guanine nucleotide exchange factor) converts GDP to GTP, which turns Ras ON
Ras needs a GAP (GTPase activating factor) to hydrolyze GTP to GDP, turns Ras OFF
describe how the MAP kinase cascade becomes activated via Ras
1) prior to signaling, Grb and SOS are bound
2) when RTK activated by a ligand, generates a phosphotyrosine residue
3) Grb2 recognizes this residue, and Grb2 activates
4) Grb2 recruits SOS
5) SOS causes Ras nucleotide exchange of GDP for GTP
Ras becomes activated
what activates the MAP cascade?
describe the MAP kinase cascade
growth factor binds from extracellular space
1) when RTK is phosphorylated, Grb2 binds to it via SH2 domain (phospho-tyrosine binding)
SOS is constituitively bound to Grb2 via its SH3 domain (which recognizes proline-rich area of Grb2)
2) SOS activates Ras
3) Ras activates Raf (MAPKKK)
4) Raf phosphorylates MEK (MAPKK)
5) MEK phosphorylates ERK (MAPK)
causes changes in protein activity and changes in gene expression
what is the relationship between factors of the MAP pathway and cancer?
MAP is a major pathway that's deregulated in cancer
RTK may be multiplied or amplified
Ras is activated by point mutations, most commonly mutated oncogen in cancers
Raf sometimes also activated- esp in melanomas
what is herceptin?
breast cancer drug
is antibody against a specific RTK that's innappropriately active in breast cancer, so interferes w/ MAP pathway
describe the PI-3 kinase pathway
1) PI-3K is a complex of a regulatory subunit, p85, and catalytic subunit, p110
2) p85 has SH2 domain that binds activated RTKs, allows access of p110 to PIP and PIP2
3) PI-3k phosphorylates PIP and PIP2 on 3-position of inositol ring (= PIP3)
4) PIP3 activate Akt, a ser/thr kinase
5) activated Akt phosphorylates substrates that promote cell survival & inhibit apoptosis
what is PTEN's function?
tumor suppressor gene
removes phosphate from 3-position of phosphoinotides (PI3)
if mutated, the PI-3K pathway will not be turned off, and cell survival continues > tumors
what are common causes of human cancer caused by mutations in RTK pathways?
1) mutations in RTKs, mutations in Ras, muations in BRaf (a type of Raf) drive pathway activation in absence of growth factors
2) mutations leading to constitutive activation of PI-3K pathway, b/c promotes cell survival
what are some similarities & differences between GPCRs and RTKs?
BOTH: interact thru phospholipase C
only GPCRs: turn on adenylyl cyclase
only RTKs: turn on MAP kinase, Akt kinase pathways
what are ligands of cytokine receptors?
prolactin, growth hormone, interferons, erythropoietin
what is similar/different btwn RTKs and Cytokine Receptors?
RTK: single polypeptide
BOTH RTK and Cytokine extracellular portion: amino terminus, binds ligands
RTK intracellular portion: carboxy terminus, intrinsic tyrosine kinase activity
Cytokine receptor intracellular portion: carboxy terminus, DOES NOT have intrinsic tyroskin kinase activity, or any other catalytic activity; non-covalently binds non-receptor tyrosine kinase that has intrinsic tyrosine kinase activity, JAK proteins
what are JAK proteins?
non-receptor tyrosine kinases associated w/ the intracellular portion of cytokine receptors
when complexes with cytokine receptor, allows cytokine receptor to behave like RTKs in that they undergo ligand-induced dimerization
describe the cytokine receptors/JAK protein complexes signal pathway
1) prolactin/interferons/growth hormone binds cytokine receptor/JAK complex extracellularly
2) cytokine receptor/JAK complex undergoes dimerization, cross-phosphorylation, activation of JAK proteins
3) JAK proteins phosphorylate intracellular portion of cytokine receptor
4) SH2-containg proteins bind to cytokine receptor
what proteins bind to activated cytokine receptor?
what does binding cause? describe pathway
SH2-containing proteins: STAT (signal transducer and activator of transcription), are transcription factors
1) activated cytokine receptors phosphorylate STAT
2) receptors dimerize when pY of one STAT binds SH2 of its dimeric partner & vice versa
3) dimerized STATs translocate to nucleus, bind to DNA, activate gene expression
what are the ligands of receptor serine/threonine kinases?
only ligands of the TGF (transforming growth factor)-beta/BMP (bone morphogenetic protein) family
what is the structure of receptor serine/threonine kinases?
Type I and Type II
both are single-pass transmembrane proteins; have extracellular ligand binding domain, intracellular ser/thr kinase domain
function of TGFbeta/BMP proteins?
embryonic development, wound healing, disease-related fibrosis, cancer
describe the signal transduction pathway for TGFbeta/BMP on receptor tyrosine kinases
1) TGFbeta ligands induce Type I and II receptors to dimerize
2) Type II receptors are constituitively active
Type II receptors phosphorylate Type I receptors
3) Type I receptor kinase activity activates
Type I receptors phosphorylate SMAD factors
4) phosphorylated R-SMADs dimerize with co-SMADs (non-phosphorylated)
5) R-SMAD/co-SMAD dimer enters nucleus, induces expression of specific genes
how is the Notch signal activated?
when a Delta or Jagged-expressing cell comes into direct physical contact w/ a Notch-expresing cell
example of contact-dependent signaling
what is unique about R-Smads?
they are a target for many kinases, so for other pathways beyond the TGF-beta/BMP signaling pathway
describe Notch pathway
1) Notch is bound by its ligand, Delta or Jagged
intracellular domain is proteolytically cleaved
2) Cleaved Notch intracellular domain (NICD) enters nucleus, interacts w/ DNA-binding factor, CSL
CSL usually inhibits Notch pathway target genes' expression
3) CSL interaction w/ NICD makes it bind w/ transcriptional co-activators, turns on expression of notch-responsive target genes
signaling also decreases Delta expression - like on/off switch
what kind of interaction does notch signaling demonstrate?
1) lateral inhibition
2) note that no amplification occurs - so need NICD to go to nucleus to have transcription activation
what are Wnt ligands?
large family of secreted proteins that have a covalently attached fatty acid
what is receptor for Wnt ligands?
describe structure of each
canonical complex of Frizzled & LRP5/6
Frizzled: 7 TM domains, bind Wnts via large extracellular N-terminus
describe canonical Wnt signaling pathway without Wnt signal
If Wnt is inactive:
B-cateninin undergoes proteolytic destruction by APC complex
APC, axin, Ck1, GSK3-beta make up complex
CK1 and GSK3-beta, ser/thr kinases, phosphorylate B-catenin, target it for proteolytic degradation by ubiquitin proteosome system
in this state, in nucleus, TCF/LEF, DNA-binding factor, inhibits expression of Wnt pathway target genes
describe the ways that Wnt signal pathway is maintained as off
when Wnt is off,
1) TCF/LEF complex binds to DNA in nucleus, inhibits txn factors from binding and expressing Wnt-related genes
2) B-catenin undergoes constant degradation
describe pathway of Wnt signaling in the presence of Wnt ligand
1) Wnt binds Frizzled-LRP5/6 receptor complex
2) axin then binds to LRP5/6, disrupting the APC complex
Dvl binds Frizzled, leads to inhibition of CK1 and GSK3-beta, they cannot phosphorylate B-catenin
3) B-catenin thus no longer exists in phosphorylated state, is stabilized
4) B-catenin migrates to nucleus, interacts w/ TCF/LEF and txn co-activators
activates expression of Wnt pathway target genes
P3000 and CBP act as transcriptional co-activators for Wnt
what is B-catenin's function, outside of Wnt pathway?
B-catenin binds intracellular region of cell-cell adhesion molecules, tethers them to actin cytoskeleton, providng stability to cell-cell junctions
keeps epithelia of epithelial cells together
central area of B-cateinin protein is where binding occurs
what does Wnt pathway regulate re: disease?
1) important in embryonic development
2) regulates stem cells in homeostatic maintenance of adult tissues
3) involved in cancer, esp colorectal cancer
describe how muations in Wnt pathway lead to polops in colon (familial adenomatous polyposis / adenomatous polyposis coli)
APC mutations means B-catenin is always produced, never degraded
pathway is locked ON
stem cells over-proliferate, develop polops in epithelial lining of gut b/c it's constantly regenerating
thus APC = tumor suppressor gene, B-catenin=proto-oncogene
what is the structure of Hedgehog ligands (HH)?
full-length protein has N-terminal signaling domain, C-terminal auto-processing domain
C-terminal domain catalyzes proteolysis of itself into N- and C-terminal halves & simultaneously attaches a cholesterol molecuel to the new C-terminis of the N-terminal signaling domain
signaling domain then has fatty acid molecule covalently added to its N-temrinus, results in mature, dually lipidated HH ligand
what is primary HH receptor?
Ptch1, patched; 12 TM protein
describe signal transduction of Hedgehog in absence of HH ligand
1) Ptch1 inhibits signaling activity of Smo, another plasma membrane protein
2) If no Smo signaling, Gli, txn factor, is phosphorylated by PKA, CK1, GSK3-beta, Gli is thus proteolytically cleaved into GliR, repressor form
3) GliR enters nucleus, inhibits HH pathway target gene expression
describe signal transduction of Hedgehog in presence of HH ligand
1) HH binds Ptch1, relieves Ptch1-mediated inhibition of Smo
2) Smo signals to prevent phosphorylation & proteolytic cleavage of Gli
3) Full-length, activated Gli protein (GliA) enters nucleus, activates expression of HH pathway target genes
what makes the Hedgehog pathway so unique?
Ptch1 is actively inhibiting Smo without the binding of any ligand
this is v different from GPCR or RTK pathways
what is significant about relationship between the primary cilium and HH signaling?
primary cilium is microtubule-based organelle, present on almost all cell types
HH signaling requires the primary cilium, most components of HH pathway localize to the primary cilium: Ptch1 is found there, Gli proteolytic cleave to GliR occurs there
In HH presence, Ptch1 exits the cilium, Smo enteres the cilium, Gli cleave stops, leading to GliA formation
what is Gorlin syndrome, what is its cause?
basal nevus syndrome
mutations in Ptch1 result in Smo signlaling, leads to constituitive HH signaling
Causes Gorlin syndrome, which causes basal cell carcinoma of skin/medulloblastoma
Ptch1 = tumor suppressor gene, then!
what do mutations in Sonic hedgehog cause?
holonprosencephaly - birth defect include cyclopia or embryonic death
cyclopamine, chemical that binds and inhibits Smo, involved