BMB - TM signalling

why signalling is needed

allow organisms to interact with environment

convey information around multicellular organisms

facilitate complex coordination

types of signalling molecules

peptide hormones

neurotransmitters

small molecules

light

gases

types of receptor proteins

enzyme coupled

GPCR

ion channel

features of signal transduction pathways

different intracellular downstream responses

signal amplification

positive and negative feedback

convergence and cross talk

NO moa

1. synthesised by NOS from L arginine

2. binds haem group in sGC

3. changes ahem orientation to planar by pulling His105 up

4. activates GC to produce cGMP

5. cGMP activates PKG causing relaxation of VSM

6. PDE breaks down cGMP

drug classes that target NO pathway

anaphylactic shock treatment, angina and hypertension treatment, viagra

treating anaphylactic shock

large drop in BP

NOS inhibitors decrease cGMP and dilation = increase BP

not used clinically due to decreased CO and death

treating angina and hypertension

reduce BP

NO providers

riociguat stimulates sGCs - used in pulmonary hypertension

nitroglycerin converted to NO by ADH

viagra

inhibits PDE to maintain cGMP and vasodilation in penile vasculature

small hydrophobic signalling molecule examples

steroid hormones

thyroid hormones

retinoids

small hydrophobic signalling molecule moa

bind nuclear receptor - DNA binding domain, ligand binding domain and transcriptionally active domain

regulatory protein holds nuclear receptor in inactive state normally

binding causes dissociation of reg protein and action as TF

eicosanoids

lipid derivatives

act locally

PGs, TXA, leukotrienes

affect inflammation and immunity

switch proteins mechanism

signal by phosphorylation

GTP binding - GTP form is active

guanine exchange and hydrolysis

RTK signal transduction

1. ligand binding causes dimerization

2. trans-autophosphorylation activates TK domains

3. further phosphorylation and docking of SH2 proteins

modular domains in signalling proteins

SH2 - pTyr

SH3 - polyproline helices

PH - binds phosphorylated lipids

MAPK cascade

series of kinases causing signal amplification

e.g. Raf - MEK - ERK

thr-X-tyr activation motif

insulin receptor signalling

1. insulin binds - autophsophorylation of C terminal tyr

2. IR phosphorylates Tyr on IRS-1

3. Grb2 binds IRS-1 through SH2

4. SOS GAP binds through SH3

5. Ras activation

6. raf MAPKA cascadr

7. Erk activates TFs by phsophorylation

conformational change of IR

activation loop moves when 3 tyr phosphorylated to make room for substrate

other examples of enzyme linked receptors

indirect TKs, ser/thr kinases, GC coupled, ethylene receptor

PTPases

specific to pTyr

CX5R motif has cys and arg which are catalytically active

phosphoryl transferred to cys then hydrolysed

PPases

non specific

PP2A - acts on metabolism, DNA replication and transcription

A su = scaffold, B = regulatory, C = catalyitc

yersinia pestis action on signalling

YopH - T3SS

ppase homologous to PTPases

promiscuous and highly active

shigella toxin

OspF inhibits MAPK

cleaves C-Ophos of threonine

phosphothreonine lyase - irreversible

cholera toxin

ADP ribosylates AC

constitutively activates leading to ionic imbalance in GI tract

heterotrimeric G protein activation

binding to activated receptor subsequent to ligand binding

activates GEF activity - convert to GTP form

dissociation of subunits

effectors of G proteins

Gs = activates AC to produce cAMP

Gi = decrease in cAMP = close calcium channels and open potassium channels, increase PI3K and PIP3 signalling

Gq = PLC activation = IP3 and DAG

Gt = activates PDIVI in response to light

adenylate cyclase activation

C2 cytosolic domain has 2 catalytic and 2 regulatory sites

2 cAMP bind per regulatory site and cause release

heterologous desensitisation of GPCRs

PKA phosphorylates

changes Gs to Gi activity

can be through any method that increases PKA activity

homologous GPCR desensitisation

GRKs phosphorylate C terminus of receptor

recruits beta arrestin = clathrin mediated endocytosis

also amplifies heterologous pathway

adrenocortical cells hormonal feedback signalling

1. ACTH produced in stress response binds GPCR on adrenocortical cell

2. cAMP activates PKA causing aldosterone secretion which acts to increase BP

3. ANF/ANP produced in response to high BP

4. binds GC linked receptor

5. produces cGMP which activates pDE to break down cAMP and prevent further aldosterone secretion

PLC pathway

1. activated by Gq linked protein

2. DAG and IP3 produced

3. DAG activates PKC

4. IP3 opens intracellular calcium stores

PI3K

phosphorylates inositol at 3 position

has adaptor and catalytic domains

PH domains bind phosphoinositide

yeast mating signalling

Mat a and mat alpha mate to become diploid through pheromones

alpha factor binds ste2 GPCR and beta-gamma recruits Mapj adaptor Ste5 and Cdc42 small G protein to activate MAPK cascade

results in activation of pheromone response elements

activation of rhodopsin/transducin system

upon light absorption, cis retinal-schiff base nitrogen covalent bond converts to trans

photoisomerisaiton

rhodopsin/transducin system - dark

1. GC makes cGMP

2. cGMP opens Na channel - influx

3. depolarisation leads to VGCC opening

4. calcium causes glutamate release

5. glutamate binds GPCRs in bipolar cells to switch off action potentials

rhodopsin/transducin - light

1. activated trans rhodopsin binds Gt - conversion to GTP form

2. alpha su regulates PDE by binding the inhibitor subunit and activating PDE

3. cGMP converted to GMP

4. Na channels close

5. hyperpolarisation - no glutamate release

adaption to light

GC is inhibited by calcium - in the dark = high calcium = inhibition of GC = less cGMP = less Na and Ca channel opening = less glutamate release

GCAP stimulates GC in absence of calcium - in light = low calcium = GC active = increase in cGMP = ion channels open = glutamate release

termination of rhodopsin/transducin system

trans retinal lysine bond rapidly hydrolysed

rhodopsin kinase phosphorylates C terminus of GPCR

GTPase activity of Gt

cone opsins

different Gt protein

3 forms of rhodopsin - red, green and blue

direct studies of GTPases

incubation with 32P labelled GTP and measure levels of 32P-GDP formed

antibodies that bind C terminus of alpha su - shows alpha required for GTPase activity

indirect studies of GTPases

ATP to cAMP conversion, radioimmuno assays, FRET, stable guanosine nts

ATP to cAMP conversion measurement

32P labelling

ion exchange chromatography

radioimmuno assays

immobilise anti-cAMP bound to radiolabelled cAMP

measure competition of antibody with cell lysate

FRET

high sensitivity

measure fluorescence of tagged PKA regulatory subunit

cAMP not bound fluorescence is different due to transfer of fluorophore to catalytic site

stable guanosine nts

non hydrolysable GTP analogues lock alpha in active state

non phosphorylatable GDP analogues prevents alpha activity

structure of Ras

G1-G5 regions for nt binding

G1-G3 contains P loop

thr35 in G2 and gly60 in G3 important for catalysis - GTP binds gamma phosphate and coordinates these residues

role of GAPs and GEFs in ras

Ras has low intrinsic GTPase activity and very stable GDP form

GAPs have arg that actis with ras lys16 - coordinates beta phosphate to stabilise transition state

oncogenic mutations in Ras

G12V in P loop and Q16N in switch region lead to low GTPase activity that can’t be accelerated by GAP

S17N dominant negative disables GTP binding

structure of voltage gated ion channels

4 domains with 6TM helices each

S4 voltage sensor - positive side chains, depolarisation causes helix to move and open channel

activation gate

selectivity pore

selectivity of ion channels

charge

water of hydration binds C=O

structure of nicotinic ACh receptor - ligand gated

pentameric - 4 TM helices per su

ACh binds between 2 exc su coordinated by C loop and trp149

hydrophobic M2 helix lines pore - ligand binding moves hydrophobic residues and pore opens, negative residues confer cation selectivity

IP3R

downstream of PLC beta and gamma - Gq and RTK

ER calcium channel

conf change - subunits twist

calmodulin structure

4 EF hands - 2 alpha helices linked by calcium binding loop

calcium binding causes conformational change in flexible linker so calmodulin can interact with target proteins

CaMKII

controlled by calmodulin

memory device

self inhibited

in presence of calcium-calmodulin = autophosphorylation activates

maintains some activity when no calcium

frequency of calcium spikes = amount of kinase activity

calcium signal termination

ER pumps

PM pumps

NCX

IP3 removal by PPases

PLC beta phosphorylated on serine by PKC and PKA to lower affinity