1
Q
What is signal transduction
A
process by which a cell responds to external factors via signaling molecules
2
Q
What is a receptor
A
a molecule on the surface or within a cell that recognises and binds with specific molecules producing an effect
3
Q
Why GPCRs are important
A
- involved in many biological processes
- very large family of proteins
- important in drug discovery
4
Q
GPCR functions
A
- senses
- cell growth
- development
- neurotransmission
- control of heart rate
5
Q
GPCR features
A
- 7 TM regions
- Extracellular N terminus
- Intracellular C terminus
- Termini differ between GPCRs
- coupe to and activate G proteins
6
Q
Family A
A
- ex = rhodopsin
- short N terminus
- ligand binds at centre of TM bundle
- Disulfide bonds between EC loops 1 and 2
- conserved proline residue in TM6
- NPXXY motif at base of TM7
- Dry motif at base of TM3
7
Q
Family B
A
- Ex = secretin
- long N terminal domain with 3 pairs of conserved disulfide bonds
- disulfide bonds between EC loop 1 and 2
- ligands initially bind with N-terminus extracellular domain via C-terminus
- Ligands are typically peptides of 20-50AA
- chalice like open structure
8
Q
Family C
A
- ex = glutamate
- venus fly trap domain
- ligands are typically small organic or inorganic molecules
- conserved disulfide bonds between EC loops 1 and 2
9
Q
General signal transduction pathway
A
- ligand binds receptor causing a conformational change
- intracellular G protein activated
- G protein interacts with adenylate cyclase converting ATP into cAMP
- cAMP activates PKA creating a cascade
10
Q
G protein features
A
- heterotrimer = a,b, y
- inactive = GDP bound to a subunit
- large relate to receptor
11
Q
G protein cycle
A
- inactive receptor and G protein bound with GDP = anchored to membrane
- ligand binds receptor causing conformational change allowing receptor and G protein interaction
- G protein binds receptor via a subunit causing a conformational change
- GTP binds a subunit
- a subunit dissociates from B/y dimer
- signal transmitted
12
Q
GaS
A
increases adenylate cyclase and cAMP
13
Q
Gai
A
decreases adenylate cyclase and cAMP
14
Q
Gaq
A
increases phospholipase C and IP3
15
Q
GPCR synthesis and forward trafficking
A
- GPCR synthesised on ribosome
- ER chaperones involved in folding and inserting GPCR
- receptor undergoes N-linked glycosylation in ER
- GPCR exits ER via copII vesicles
- GPCR enters ER-golgi intermediate compartment via Rab1 and 2 proteins
- receptor transports through golgi where it matures using Rab6 GTPase
- if quality control fails then it is recycled back to ER
15
Q
GPCR regulatory processes
A
- phosphorylation
- desensitisation
- internalisation
- recycling
- degradation
16
Q
Splice variants
A
- altered mRNA splicing
- > 1 exon needed (Family B and C)
- can lead to truncated receptors and variable domains/loops
- can alter binding, signalling and expression
17
Q
Sequence variants
A
- polymorphisms and receptor mutants
- change a single AA
- can cause gain or loss of function
18
Q
GPCR loss of function mutants
A
- intracellular retention
- loss of agonist binding
- loss of intramolecular activation
- loss of G protein binding and interactions
19
Q
GPCR gain of function
A
- constitutive activation
- increased activity of ligand
20
Q
Glycosylation
A
- addition of oligosaccharides to specific AA in extracellular portions
- N-linked = in ER, modified in golgi, acceptor site = NxS/T
- O-linked = in golgi, acceptor sites = S/T
- required for cell surface expression of some GPCRs
- conflicting data on ligand binding
21
Q
Palmitoylation
A
- addition of palmatite to cysteine in intracellular domains via thioester bonds
- can be constitutively active important for expression = anchors part of tail in membrane creating a functionally important 8th helix
- can be dynamic = mask or unmask interaction sites
- dynamic can be induced via agonists
22
Q
Phosphorylation
A
- can uncouple G proteins via phosphorylation of S/T residues in ICL/C-terminus
- many different kinases involved
- 2nd messenger dependent PKA/PKS can phosphorylate causing desensitisation
- GRKS = families which have different tissue distributions, can increase affinity for arrestin = decreased signalling
- different kinases in different tissues give different phosphorylation barcodes = different functions
23
Q
Ubiquitination
A
- reversible addition of Ub to lysine
- occurs in intracellular domains of GPCRs with or without ligand activation
- important for internalisation
- can occur in arrestin which increases degradation fate
- during biosynthesis it can mark misfolded GPCRs to degradation
- may modify signalling via masking or exposing interaction sites
24
Consequences for heterodimerisation
- altered ligand binding
- coupling to a new G protein
- Increased or decreased G protein coupling
- Switch from G protein to B-arrestin coupling
- Receptor expression
25
GABA b receptor
- dimer of R1 and R2
- obligatory heterodimer
- R1 = ER retention motif, can bind GABA, cannot signal
- R2 = cell surface expression, cannot bind GABA, can signal
- binding of GABA to R1 causes a conformational change in R2 allowing for signalling
- R2 masks ER motif in R1 allowing cell surface expression
- Depolarisation assay shows that they are an obligatory dimer
- cellular experiments shows co-expression
26
a1B and a1D adrenergic receptor dimers
- 1B is expressed at cell surface alone but 1D is retained intracellularly
- when co-expressed then they are both expressed at cell surface
27
Taste receptors
- different combinations of receptors generates dimers with different ligand specificities
- T1R2 + T1R3 = sweet
- T1R1 + T1R3 = umami
- knockout mice of certain receptors showed loss of detection of specific tastant molecules
28
GPCR modulating binding affinity - opiod receptors
- when Kappa and delta receptors dimerise they are no longer selective to their specific ligands
- when mu and delta receptors dimerise they can recruit arrestin but cannot individually
29
Dopamine receptors - alter G protein coupling
- D1 = Gs
- D2 = Gi
- dimerisation of D1/D2 couples Gq
30
Methods to detect dimerisation
- immunoprecipitation
- RET based methods
- heteromer selective antibodies
- heteromer specific ligands
31
Basis of RET
transfer of energy between a donor and acceptor when they are closer than 100A
32
BRET
- donor and acceptor proteins are attached to 2 different GPCRs
- donor = Rluc
- acceptor = YFP
- if GPCRs dimerise in the presence of substrate coelenterazine then energy will be transferred and YFP will emit light
33
FRET
- Donor = CFP
- Acceptor = YFP
- CFP is excited by a certain wavelength of light causing energy transfer to YFP
34
Time resolved FRET
- donor = europlum
- acceptor = alexa or APC
- acceptors are attached to receptor specific antibodies or ligands for the receptors
35
BiFC
- fluorescence protein is split into 2 parts with each part fused to a different receptor
- if receptors dimerise then a functional fluorescence protein forms = fluoresce
36
Functions of accessory proteins
- expression
- binding
- signalling
- regulation
37
MRAP characteristics
- Has 2 splice forms MRAPa and MRAPB
- Single TM proteins
- form anti-parallel homodimers
38
MC2R
- poorly expressed at cell surface in noradrenal cells
- mutations cause FGD
- MRAP has association with FGD
39
MRAP2 characteristics
- orthologue of MRAP
- single TM
- also a dimer
40
MC2R and MRAP
- alone MC2R is found in ER
- when co-expressed with MRAP then it is expressed at the cell surface and shows increased cAMP signalling
41
MC1R/MC3R and MRAP
- receptors are normally expressed at cell surface
- when co-expressed with MRAP there is decreased signalling via unknown mechanisms and no effect on expression
42
MC4R/MC5R and MRAP
- receptors are normally expressed at cell surface
- when co-expressed with MRAP then transport from ER is blocked = decreased expression and signalling
43
RAMP characteristics
- family of 3 proteins
- Single TM
- long extracellular N terminus
- Short intracellular C terminus
44
CGRP receptor
- heterodimer of CLR and RAMP1
- 37AA
- expressed in trigeminovascular system
- expression increased during a migraine attack
- believed to be involved in pain and vasodilation symptoms
45
CLR and RAMP1 cell surface expression
- not expressed when alone
- RAMP1 has an ER retention motif
- when co-expressed = expression at cell surface = increased cAMP signalling
46
RAMPs altering ligand specificity of CLR
- RAMP1/CLR binds CGRP
- RAMP2/3 with CLR bind AM
- RAMP1 and RAMP2 are structurally similar but differ by a single amino acid dictating ligand binding
- RAMP1 = tryptophan
- RAMP2 = phenylalanine
47
Gepant migraine drugs
work by blocking CGRP binding via binding of both CLR and RAMP1
48
RAMPS affecting trafficking of CLR from cell surface
- RAMP2 = targets CLR for internalisation and degradation
- RAMP3 = recycling endosome
49
VPAC1 and RAMP2
increased IP signalling in a ligand dependent manner
50
G protein a subunit structure
- GTPase domain
- helical domain which interacts with B subunit
51
Conformational change in GPCR allowing for G protein interaction
- DRY motif in TM3 is altered so ionic lock between TM3 and TM6 Is broken
- TM6 can move outwards by 14A
- TM5 extends by 2 helical turns
- Movement accomodates for G protein a5 helix
- change in NPxxY motif in TM7 allowing for receptor activation
52
Role of RGS
regulate G protein activity by interacting with the a subunit to promote the transition state for GTP hydrolysis = promotes inactive form
53
B-arrestin characteristics
- contain many protein interaction motifs such as clathrin binding
- have high affinity for phosphorylated GPCRs
- involved in many different functions = internalisation, signalling and desensitisation
- can adopt many different conformations
54
B-arrestin interactions with GPCRs - B-arrestin conformational change
- when arrestin interacts with GPCR the arrestin undergoes a conformational change as the polar core is disrupted
- C terminal tail becomes exposed, exposing clathrin and adaptor protein binding sites
- clathrin coated vesicles can be recruited = GPCR endocytosis
55
B-arrestin interactions with GPCRs - GPCR conformational change
- changes similar to that of G protein interaction
- TM6 moves less ~10A
56
B-arrestin shape shifting
- different phosphorylation can cause different conformational change
- different protein recruited = different response
57
B-arrestin as a scaffold for signalling
- scaffold for MAP/ERK, Janus kinase and AKT signalling pathways
- SiRNA KO models of B-arrestin in mice showed a decreased rate in pERK via angiotensin 1 and PTH 1 receptors
58
What type of proteins are GIP
intracellular proteins
59
Biased receptor
- receptor biased for a specific signalling protein
- receptor activated by a ligand is in a conformation specific to a signalling protei
60
Biased ligand
each ligand generates a different conformation activating a different pathway
61
Intracellular protein bias
intracellular proteins can affect receptor conformation thus altering which ligand can bind
62
Cell type or system bias
- not strictly bias
- proteins expressed can be exclusive to cell type so activate different pathways thus appearing biased
63
Biased agonist PAC1 receptor
- 2 peptides = PACAP38 and PACAP27
- PACAP38 activates both cAMP and pERK1/2
- PACAP activates cAMP but not pERK1/2 = BIASED
64
Biased agonists - beta blockers
some beta blockers antagonise cAMP signalling but activate pERK1/2
65
Why is bias an important consideration for drug safety and screening
allows for detection of more drugs and potential side effects
66
Measurement of arrestin recruitment/activation for screening
done via either energy transfer or a complementation approach
67
Label free technology - drug screening
- multi probe screening approach which measures all pathways in the cell
- measured via wavelength shift
- wavelength differs by agonist, receptor and cell type
- problem = picks up everything so can be hard to distinguish
68
Types of GIPs that regulate GPCR function
- G proteins
- Arrestins
- Signalling kinases = JAK2
- scaffold proteins = NHERF
69
3 roles of GIPs
- GPCRs can signal directly via GIPS
- can enhance G protein signalling
- can promote recycling or degradation of GPCRs after endocytosis
70
GPCRs can signal directly via GIPS - Angiotensin I receptor
- angiotensin II binds angiotensin I receptor = activation
- results in JAK2 directly binding
- JAK2 associates with SHP2/PTPNII = JAK2 signalling activation
- STAT phosphorylated = transcription activation
71
GIPs enhance G protein signalling - PTHI receptor
- some cells preferentially couples to GaS = cAMP signalling
- some cells express NHERF which causes Gaq signalling due to recruitment of downstream proteins
72
GIPs promote degradation of GPCRs - u-opiod receptor
- normally recycled after interaction with B-arrestin
- when GASP1 is expressed the receptor is targeted for lysosomal degradation
73
GIPs promote recycling of GPCRs - B2 adrenergic receptor
- normally receptor is degraded after internalisation
- when NHERF is present then it is recycled
74
Methods for tracking GPCRs
- fluorescent ligands
- fluorescent antibodies (primary and secondary, or primary with fused fluorescence tag)
- fusing fluorescent proteins to GPCR
- can all be combined with markers such as antibodies or dyes to locate intracellular compartment
75
Epiptope tags
- attached to GPCR to be recognised by specific high affinity antibodies for identification
- solves problem of antibodies usually being low affinity and non-specific
- ex = HIS tags
76
Marker examples
- EEA1 antibody = early endosome
- calnexin = ER
- DAPI dye = nucleus
77
2 types of lipid raft microdomains
- planar lipid raft
- caveolae
78
Planar lipid raft characteristics
- continuous with plane of the membrane
- promotes clustering of proteins
79
Caveolae characteristics
- create invaginations in membrane
- 25-100nm diameter
- cholesterol and sphingomyelin rich assemblies
- contain caveolin protein
- meeting point for cell signalling proteins
80
Caveolae mechanisms
- can prevent receptors from having easy access to its ligand = no signalling
- can cluster signalling proteins = promotes signalling
- caveolin scaffolding domain promotes clustering
- can promote crosstalk between different receptors
81
Scaffolds (lipid raft microdomains) changing specificity and activity
- can change the function of a protein that bound by causing a conformational change
- cytoskeletal proteins may help scaffold and organise
82
B-arrestin mediated endosomal signalling
- during internalisation clathrin is removed by B-arrestin remains attached
- B-arrestin is active and free to bind nearby signalling proteins = signalling occurs
- either causes degradation or recycling
83
Adenylate cyclase mediated endosomal recycling - TSH receptor
- TSHR at cell surface is activated via TSH
- GaS couples = binds adenylate cyclase = cAMP signalling
- receptor is internalised and travels to trans-golgi network
- receptor is still bound to GaS and adenylate cyclase = continued sustained signalling
84
Signalling in breast cancer - CXCR4
- in normal cells CXCR4 has a high affinity for the Gai signalling = no activation of G12/13
- In triple negative breast cancers then G12/13 is upregulated = CXCR4 activates it = metastasis
85
Viruses in cancer - KSAV
- encodes for vGPCR oncogene
- GPCR is constitutively active and is the closest human homologue to CXR1 and CXR2
- Active due a mutation in TM3 from DRY to VRY so TM3 and TM6 cannot interact = always in active conformation
- activates G12/13 and Gaq pathways = release of cytokines = angiogenesis in a paracrine manner
86
GPCR mutation causing cancer - TSHR
- associated with thyroid cancer
- cluster of mutations on or around TM6
- mutations disrupt interactions between TM3 and TM6 = constitutively active
87
Receptors involved in insulin release and secretion
- GLP1-R = GaS signalling = activates PKA = insulin release
- GHSR = Gai signalling = inactive PKA = inhibit insulin release
- M3R = Gaq signalling = release of DAG and IP3, DAG activates PKC and IP3 induces Ca release = both stimulate insulin release
88
Integration of GPCRs and glucose transporters in insulin release regulation
- GLUT2 and IGF1
- signals from GPCR integrate with signals from these transporters
- K+ channel blocked = K+ accumulates in cell causing depolarisation = increased Ca influx = stimulates insulin release
89
Diabetes drugs
- K+ channel blockers
- GLP-1 mimetics
- work to increase GaS signalling = stimulate insulin release
90
B-arrestin2 KO mice models - morphine and opioid
- used to study u-opioid receptor
- WT = morphine activates receptor causing pain relief but some negative side effects
- KO = morphine activates receptor causing enhanced pain relief and fewer side effects
- suggests that B-arrestin2 signalling involved in negative side effecs
- used these results to design a G protein biased ligand which activates Gai but not arrestin
91
Arrestin knock in mouse models
- WT = normal GPCR which couples to both G proteins and arrestin
- KI = modified receptor which lacks phosphorylation sites so arrestin cannot bind = G protein bias
- Advantage of only needing to change a single receptor wheras KO models need to change all receptors
92
Receptor bias
receptor has conformation that is bias for one signalling protein thus causing a specific signalling pathway
93
Ligand bias
different ligands binding to the same receptor cause different signalling pathways due to different conformations
94
Intracellular protein bias
intracellular protein can influence which ligand is bound to the receptor
Biosci 353
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