Transmembrane Signalling Flashcards
needfor cell signalling
- interact with environment
- intercellular signalling for in a multicellular organism - facilitates coordination eg of growth control
- central regulation of metabolic functions of different tissues
cAMP and dictyostelium
unicellular form - release cAMP upon starvation - chemoattractant, enables aggregation into multicellular form
endocrine
specific organ/ gland secretes specific hormone into circulation which acts on distant cells
short range signals
paracrine - eg NO, GFs, cytokines
autocrine - amplify incoming signal
gap junctions and signalling
signals transmitted directly through pores in membrane
discriminating between different external signals
specificity and expression of receptors
signalling pathways activated by a particular receptor
common features of signal transduction pathways
- amplification
- specificity
- adaptation
- integration
- modularity
lipid soluble hormones
diffuse into the cell, bind specific cytoplasmic receptors, translocate to the nucleus, the receptor-hormone complex interacts directly with the target DNA
second messenger
molecules that relay signals from cell-surface receptors to target molecules inside the cell
eg cAMP, DAG, Ca2+
changes in second messenger levels correlate with physiological effects of the first messenger, need a removal method
general model for a signalling pathway
- specific receptor for the stimulus
- transduction mechanism to transfer info from outside to inside, often uses amplification
- effector system, eg enzyme generating second messenger, helps amplification
- response element - eg protein kinase, delivers info to final target (ampl)
- mechanism of signal termination
switch proteins
active, inactive states
G proteins, phosphorylated proteins
structural feature of signalling proteins
many protein-protein interaction domains
scaffold/ adaptor proteins
have multiple specialised domains which act as docking sites for other proteins
so can form large protein complexes
Grb2
an adaptor protein with 2 SH3 and a SH2 domain
NO signalling
local signal as is unstable
- production by vascular endothelium, diffuse to vascular smooth muscle, stimulates guanylyl cyclase activity which converts GTP to cGMP, triggering relaxation - vasodilation
- NO synthase is a calcium dependent enzyme
molecular action of NO on guanylyl cyclase
NO binds to the haem of GC, restoring a planar structure and causing a protein conformational change via movement of an attached histidine
pharmacology + the NO system
block NOS activity with arginine analogues: eg L-NAME - treat anaphylactic shock via increasing blood pressure (prevent vasodilation)
- viagra - blocks cGMP PDE5 , causing increased NO levels and sustained vasodilation in erectile tissue
3 steroid hormones
cortisol - stress - adrenal gland
- oestrogen, testosterone - sexual dev
- thyroid hormone - metabolism
nuclear receptor superfamily
TFs with ligand binding, DNA binding and transcriptional activation domains
glucocorticoid receptor
binds Hsp90 wo ligand (so inactive)
glucocorticoid binding displaces Hsp90 - enables binding to regulatory DNA sequences - associate with HAT for expression of target genes
thyroid hormone
absence of hormone - TH receptor is associated with a corepressor. hormone binding results in activation of transcription
roles of water-soluble hormones
maintain homeostasis, respond to external stimuli eg fight or flight, follow cycic/ deelopmental programmes (eg sex hormones)
examples of water soluble hormones
adrenaline, NA
peptide hormones
neurotransmitter
carries signals between neurones or from neurones to target cells
eicosanoids
lipid signalling molecules - leukotrienes, prostaglandins, thromboxanes
derived from arachidonic acid - from phospholipids
producing eicosanoids
leukotrienes - lipoxygenase pathway
prostaglandins, thromboxanes - by cyclooxygenase pathway
hydrolysis of membrane lipids - by phospholipases
cyclooxygenase
enzyme of pathway to generate prostaglandins and thromboxanes
target of antiinflammatory drugs - aspirin, NSAIDs - reducing inflammation and pain
calcium and phospholipase A2
binding of calcium to PLA2 promotes its translocation from the cytoplasm to the membrane. cPLA2 then phosphorylated by MAPKs
6 types of receptors
- GPCRs
- RTKs
- receptor guanylyl cyclase
- gated ion channel
- adhesion receptor (integrin)
- nuclear receptor
adhesion receptors
cell adhesion initiates intracellular signalling pathways which regulate other aspects of cell behaviour. eg GFs - cytoskeletal rearrangements resulting in cell movement/ shape changes
integrins - receptors for cell attachement to ECM, also interact with cytoskeleton
integrins
integrins - receptors for cell attachement to ECM, also interact with cytoskeleton
1 a and 1 B domain, eith a cytoplasmic domian (int w cytoskeleton), TM domain, extracellular domain
inactive - extracellular domain is folded
contact extracellular ligand - extracellular domain straightens, cytoplasmic tails move apart altering their interactions with intracellular proteins eg actin cytoskeleton
binding of integrins to ECM ..
activation of FAK (focal adhesion kinase)
phosphorylation of FAK - binding of signalling molecules eg the Grb2-Sos complex
activation of Ras, PI3K, PLCy.
3 types of protein kinase
Ser/ Thr
Tyr
Thr/ Tyr (unusual)
effects of protein phosphorylation
- alter local charge density (insert negative charge)
- alter shape as well as local charge density
==> change in protein activity and capacity for interaction with other proteins
how does protein phosphorylation enable diversification of signalling pathways?
kinases are not specific to 1 substrate, pathway branches
protein kinase specificity
depends on primary sequence surrounding the target amino acid, some kinases have consensus sequences while some have broad specificity
RTK structure
extracellular ligand binding domain, single TM a helix, cytosolic domain with Tyr kinase activity
RTK activation mechanism
ligand binding - dimerisation - autophosphorylation
experiment on RTK activation mechanism
insulin receptor = already a dimer
produce chimeric receptor with insulin R extracellular domain and EFG TM/ cytosolic domain - could only transmit signals when occupied by insulin, so dimerisation was not sufficient for activation
effects of RTK autophosphorylation
receptor activated to phosphorylate other substrates
pY residues act as a template to bind SH2 domains of other proteins (receptor forms signalling complex - colocalisation of molecules allows interactions)
how can receptor phosphorylation act as a second messenger?
signalling proteins bind phosphorylated tyrosines via their SH2 domains
the catalytic activity of the clustered proteins act on substrates in the vicinity of the receptor kinase eg membrane lipids
eg PI3K, Ras
MAPK general activation
Downstream of RTK…
MAPKKK=ser/thr kinase, phosphorylates MAPKK, a Tyr/Thr kinase which phosphorylates and activates MAPK
downstream of MAPK are more kinases: MAPKAPs which regulate gene expression. eg JNK, p38 in the stress response
highly directed while highly diverse (branched) signal transduction
EGFR MAPK pathway
EGF - receptor dimerisation and autophosphorylation
Grb2 binds EGFR pY via its SH2 domain
Sos (Ras-GEF) binds Grb2 SH3 domain
Sos activates Ras (membrane-bound) by promoting exchange of GDP for GTP
Active Ras-GTP activates Raf (MAPKKK)
Raf activates Mek (MAPKK)
Mek activates Erk (MAPK)
Insulin receptor - general
uses same principle as other RTKs but only recruits insulin-receptor substrate 1 - which becomes phosphorylated and acts as a scaffold for other proteins
insulin receptor - structural changes
IR = a dimer of aB monomers, the a subunits bind insulin and the B subunits have the protein kinase activity
insulin binding activates this PK activity
each B phosph 3 Tyr on the other B
the autophosphorylation opens up the active site: movement of the activation loop makes room for the target protein in the substrate binding site
insulin receptor signalling pathway
- IR binds insulin, autophosphorylates
- IR phosphorylates IRS1
- SH2 of Grb2 binds pY. Sos binds Grb2, then Ras, converted to active GTP-Ras form
- Ras activates Raf
- Raf activates Mek
- Mek activates Erk
Erk moves into the mucleus, phosphorylates nuclear TFs eg Elk1 is activated - phosph Elk1 joins SRF to stimulate transcription of genes needed for cell division
Indirect protein tyrosine kinases
cytosolic domains do not have catalytic activity, ligand binding causes dimerisation and cross-phosph of associated non-receptor protein tyrosine kinases
example of a non-receptor tyrosine kinase
c-Src
of interest as the pathway is overactivated in many tumours
regulation of c-Src - autoinhibition
an inhibitory Tyr of C terminus is phosphorylated under resting conditions, C terminus folds back and interacts with c-Src’s SH2 domain: obscuring the SH1 catalytic site
3 groups of protein phosphatases
- non-specific (acid, alkaline phosphatases)
- phosphoserine/ threonine specific
- phosphotyrosine specific
protein tyrosine phosphatases
phosphatase domain has 11 residue signature sequence called CX5R motif - contains essential Cys, Arg residues
covalent Cys-phosphate intermediate - then hydrolysed
protein ser/thr phosphatases
eg PP2A: regulation of metabolism
heterotriimer with scaffold, regulatory and catalytic subunits
guanylyl cyclases
extracellular ligand binding domain, single TM a helix, cytosolic catalytic domain
Notch
cleaved when receptor is activated by delta ligand, can directly modify transcription
ethylene receptors - plants
empty receptor is active - without ethylene, the empty receptor activates a kinase which shuts off ethylene responsive genes. when ethylene is present, the receptor and kinase are inactive and the genes are transcribed
– relief of repression is a common mechanism in plants
in which cell types are ion channels particularly important
nerve, muscle
key properties of ion channels
rapid transport
can be highly selective
nerve impulse and ion channels
AP travels along axon - membrane depolarises, Vm changes from -60mV to +30mV. due to rapid opening and closing of VG Na+, K+ channels
ligand gated ion channels
open in response to binding of neurotransmitters/ other signalling molecules
VG ion channels
open in response to changes in PM electric potential
ligand gated ion channels
example
multisubuni - dif families have 3-5 subunits (can be homo or hetero)
nAChR - pentameric, each subunit has 4 TM a helices
P2X family - trimeric
nAChR
ACh binding causes conf change which is transduced to the membrane domain
a helices lining the pore relax, removing the hydrophobic girdle blocking the channel and allowing ion flow
structure of VG ion channels
Na+, Ca2+ - single polypeptide with 4 homologous domains, each containing 6 TM a helices
K+ - 4 separate polypeptide chains
which helix of a VG ion channel is a voltage sensing helix
S4 - contains many + amino acids, so moves upon depolarisation - causing a conformational change which opens the channel
ion selectivity in K+ channels
pore is lined with C=Os - displace hydration shell of K+ so K+ can pass through
Na+ is too small to interact and remains bound to water
calcium signalling
intracellular Ca2+ is very low - use intracellular and extracellular Ca2+ as a second messenger - Ca2+ channels open upon dif stimuli
eg upon membrane depolarisation - leads to neurotransmitter exocytosis
IP3
binds ER receptors, opens Ca2+ channels
IP3 = from PIP2 hydrolysis by PLC
PLC
2 forms - 1 stimulated by GPCRs, 1 stimuated by tyr kinases
PLC action
cleavage of PIP2 (a membrane phosphoinositol/ lipid) into IP3 (remains in membrane) and DAG (released to cytoplasm)
experimental dissection of signalling pathways
- protein-protein ints
- probe roles of specific Tyrs with alanine screening
- order proteins in pathway - genetic screen, mutant rescue
elemental event - IP3/ IP3R
random channel opening - may trigger opening of adjacent IP3R and rise in Ca2+: spark/ waves
IP3R
present on ER membrane
opening leads to Ca2+ release
global opening of IP3Rs
need sufficient external stimulus, causes sustained rise in intracellular Ca2+
IP3 signal termination
phosphatases remove phosphate from IP3, giving inositol
Ca2+ is exported from the cytoplasm - pumping into ER via SERCA, Na+Ca2+ exchangers and high affinity plasma membrane pumps
PLCB phosphorylated by PKA, PKC - lower enzyme activity
downstream effects of calcium
actions mediated via calmodulin (CaM) which binds Ca2+ via its EF hand domains, causing a conformational change which allows CaM to interact with target proteins ... NO synthase MLCK adenylate cyclase PMCA - plasma mem Ca2+ pumps
downstream effects of PLC/ IP3 pathway
activate cellular activity/mitogenesis
PI3 kinase
phosphorylates PIP2 to produce PIP3
PI3K is a dimer with a regulatory and catalytic subunit - modular structures which can interact with other molecules too.
activated by GPCRs, intracellular tyrosine kinases
effectors - Rac, a small GTP binding protein
alter cytoskeletal function - cell motility, vesicle trafficking, DNA synthesis
GPCRs
highly conserved
alternate between 2 discrete conformations as ligand binding causes a conformational change to active state, allowing binding of heterotrimeric G protein to cytoplasmic face of receptor
ligand binding to GPCR
ligand recognises binding pocket - which can be formed by external loops or deep within the cluster of transmembrane helices
initiates transmission of conformational change to intracellular domains of molecule - 5th and 6th helices are key to this signal transduction
examples of GPCRs
receptors for peptide hormones, glycoprotein hormones, some neurotransmitters, amines, nucleotides, eicosanoids
experiment which shows receptor mobility
(not definitive evidence!) - fusion of cell containing adenylate cyclase but not B-AR with cell containing B-AR but not adenylate cyclase - cell responsedto adrenaline by increasing cAMP. so B-AR or adenylate cyclase must be able to move in the membrane
though this is not good evidence as there could be movement of a cytoplasmic signal between the 2 proteins
desensitisation/ adaptation of GPCRS - 2 mechanisms
- PKA phosphorylates C-terminus of receptor - blocks capacity to bind to Gs but allows interaction with Gi - causing signal termination
= heterologous desensitisation as any ligand that activates adenylate cyclase will have this effect. (dual effect as also turning ON an inhibitory pathway) (binary - all or nothing)
activity of a specific protein kinase - BARK - which phosphorylates a different site of the receptor which is only accessible in active receptors (dose-dependent), allowing recruitment of B-arrestin, an inhibitory molecule, blocking signal transduction through BAR - homologous desensitisation
Gs
stimulatory, increases adenylate cyclase
a, B, y subunits
Ga has a ras-like G domain and a helical domain
GB has a helical domain and a B propeller
GB and Gy are tightly associated and act as a single unit
How do GPCRs transduce signals?
ligand binding to extracellular domains transmits a conformational change to the cytosolic domain, allowing association with a specific G protein.
binding of the G protein promotes GDP for GTP exchange, leading to dissociation of the G protein from the receptor and dissociation of the a subunit from the By subunit
G protein families
high level of conservation in GTP binding site
Gs, Gi and Gq like a subunits
responses in which G proteins are involved
adrenergic receptor activation
heart rate and contractility increase BP increase reduced blood flow to peripheral organs bronchodilation inhibition of insulin release stimulate glycolysis and glycogenolysis
a1
a2
B1/B2
a1: Gq, phospholipase C
a2: Gi, inhibit adenylate cyclase
B1/B2: Gs, activate adenylate cyclase
adenylate cyclase
9 dif enzymes of similar structure,
2 TM domains separated by a cytoplasmic loop which is the regulatory and catalytic region.
convert ATP to cyclic AMP
regulators of adenylate cyclase
Gsa, Gia, PKCa, CaM
effects of cAMP
affects activity of PKA
PKA
a tetramer of 2 regulatory and 2 catalytic subunits - the catalytic subunits dissociate upon binding of cAMP to each regulatory molecule
Measuring adenylate cyclase activity
conversion of 32P radiolabelled ATP to cAMP - separate using ion exchange chromatography
radioimmuno/ binding protein assay - displace radiolabelled cAMP from antibody using test sample (whole cell extract)
fluorometric measurement - fluorescence of tagged PKA regulatory subunit changes upon binding cAMP (microinject)
cAMP breakdown
by phosphodiesterases
can be regulated eg by protein phosphorylation, Ca2+/CaM
cyclic AMP PDE
activated by cGMP
= cross talk: cAMP dependent processes can be modulated by ligands which activate cGMP production
ACTH cAMP PDE example
ACTH stimulates aldosterone production by the adrenal cortex in a cAMP dependent manner. this causes an increase in blood volume. to limit this increase, atrial natriuretic factor is synthesised in the heart and leads to cGMP generation by activating receptor adenylate cyclase. cGMP binds and inactivates cAMP PDE, preventing PKA activation and turning off aldosterone production.
thrombin - protease activated receptor
PAR1 is a receptor for thrombin, a serine protease. thrombin clips off a short peptide from the receptor, revealing a new N terminus which can bind and activate the receptor.
the receptor therefore contains a tethered ligand. synthetic peptides of the same sequence stimulate the receptor without cleavage. eg TRAP - thrombin receptor activatory protein
Phospholipase C - activation by G proteins
leads to production of 2 second messengers - IP3 and DAG
DAG
IP3
diacylglycerol - activates PKC
IP3- binds IP3R, leads to release of calcium from ER, CaM activates protein kinase C
protein phosphorylation - response
photodetection in the eye - general
retinal cells deliver impulse to optic sensory neurones upon fall in internal calcium - which is regulated by cytoplasmic GMP
rod and cone cells
rod cells - black and white vsion
cone cells - red, green or blue sensitive iodopsin
perceive light, send signals to the brain, rapid activvation and termination, adapt to changes in ambient light
cGMP = second messenger
Rhodopsin
a G protein linked photoreceptor in the membrane of the rod outer-segment disc
retinal = chromophore - covalently linked within rhodopsin
light absorption converts cis retinal to the all-trans form - the conformational change activates the receptor
so retinal is the ligand for rhodopsin and the ligand only adopts an activatory conformation upon absorption of light.
Rhodopsin pathway
- light activates retinal, which activates Rhodopsin - the receptor
- activated rhodopsin binds transducin, a heterotrimeric G protein - causing exchange of GDP for GTP, By subunit dissociation
enables Gta to activate its effector, cGMP PDE (via Gta binding an inhibitor PDE subunit)
PDE converts cGMP to GMP so cGMP levels fall
falling cGMP levels causes closure of cGMP gated cation channels, causing hyperpolarisation
what does falling cGMP levels cause in phototransduction
closure of cGMP gated cation channels
hyperpolarisation
dark current
phototransduction
cell is partly depolarised due to influx of Ca2+ and Na+ eg via cGMP gates Na+ channels
cGMP PDE not activated
why does phototransduction signalling start rapidly
activated rhodopsin can activate many Gts, Ca2+ channels bind 3 cGMP cooperatively
termination of phototransduction signalling
trans retinal is rapidly hydrolysed and uncoupled from the receptor
rhodopsin kinase rapidly phosphorylates the receptor, which then associates with arrestin so cannot bind Gt (homologous desensitisation)
Gt has GTPase activity so inactivates
adaptation in phototransduction
cGMP and Ca2+
guanylate cyclase is inhibited by Ca2+ so activity rises when Ca2+ internal falls, generating cGMP which can help return the channels to their open state (mediated by guanylate cyclase activating protein)
Ca2+ entry and cGMP formation establish an equilibrium - adaptation - allows eye to function over wide range of ambient light levels
steady state is disrupted by altered PDE activity/ rhodopsin desensitization
GCAP
guanylate cyclase activating protein
binding Ca2+ inactivates
colour vision
cone cells
dif forms of rhodopsin - different environment for retinal chromophore, different absorption spectra
eg vertebrates have 3 forms of rhodopsin which respond to red, green and blue light
studying the G protein cycle - stable guanosine nucleotide analogues
non-hydrolysable analogues of GTP - a subunit becomes locked in a permanently active state
stable analogue of GDP prevents G protein activity
ALF4- structure matches that of PO42-, triggers release of By subunits as achieved by GTP
inactivation of GTPases by covalent modification
cholera toxin acts on Gs and prevents GTP hydrolysis so the a subunit is constitutively active
By sununits
also have signalling roles
processes involving small GTPases (monomeric)
nuclear transport, cytoskeletal rearrangement, cell growth, differentiation, adhesion
Ras
(RAt Sarcoma)
viral oncogene
activates TFs, controls gene expression
mutated forms are heavily implicated in cancer, with most mutations involved in the guanosine binding region
also undergoes a GTP/ GDP cycle however has low catalytic activity/not able to hydrolyse GTP to GDP quickly
Ras structure
5 conserved regions
GTP binding region
switch regions - move upon GTP binding
effector domain interacts with downstream targets
Ras pathway
isolated ras is catalytically inactive
GTP hydrolysis requires the action of GAPs
guanosine exchange is by GEFs
active in GTP bound form due to movement of switch regions
GAPs/ GEFs work by inserting residues into active site
oncogenc ras mutations
low rate of GTP hydrolysis - constitutively active
