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Biosci 353 > Test 2 > Flashcards

Test 2 Flashcards

1
Q

What is signal transduction

A

process by which a cell responds to external factors via signaling molecules

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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

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3
Q

Why GPCRs are important

A
  • involved in many biological processes
  • very large family of proteins
  • important in drug discovery
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4
Q

GPCR functions

A
  • senses
  • cell growth
  • development
  • neurotransmission
  • control of heart rate
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5
Q

GPCR features

A
  • 7 TM regions
  • Extracellular N terminus
  • Intracellular C terminus
  • Termini differ between GPCRs
  • coupe to and activate G proteins
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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
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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
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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
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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
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10
Q

G protein features

A
  • heterotrimer = a,b, y
  • inactive = GDP bound to a subunit
  • large relate to receptor
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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
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12
Q

GaS

A

increases adenylate cyclase and cAMP

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13
Q

Gai

A

decreases adenylate cyclase and cAMP

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14
Q

Gaq

A

increases phospholipase C and IP3

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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
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15
Q

GPCR regulatory processes

A
  • phosphorylation
  • desensitisation
  • internalisation
  • recycling
  • degradation
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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
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17
Q

Sequence variants

A
  • polymorphisms and receptor mutants
  • change a single AA
  • can cause gain or loss of function
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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
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19
Q

GPCR gain of function

A
  • constitutive activation
  • increased activity of ligand
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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
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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
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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
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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
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24
Q

Consequences for heterodimerisation

A
  • 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
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25
Q

GABA b receptor

A
  • 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
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26
Q

a1B and a1D adrenergic receptor dimers

A
  • 1B is expressed at cell surface alone but 1D is retained intracellularly
  • when co-expressed then they are both expressed at cell surface
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27
Q

Taste receptors

A
  • 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
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28
Q

GPCR modulating binding affinity - opiod receptors

A
  • 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
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29
Q

Dopamine receptors - alter G protein coupling

A
  • D1 = Gs
  • D2 = Gi
  • dimerisation of D1/D2 couples Gq
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30
Q

Methods to detect dimerisation

A
  • immunoprecipitation
  • RET based methods
  • heteromer selective antibodies
  • heteromer specific ligands
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31
Q

Basis of RET

A

transfer of energy between a donor and acceptor when they are closer than 100A

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32
Q

BRET

A
  • 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
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33
Q

FRET

A
  • Donor = CFP
  • Acceptor = YFP
  • CFP is excited by a certain wavelength of light causing energy transfer to YFP
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34
Q

Time resolved FRET

A
  • donor = europlum
  • acceptor = alexa or APC
  • acceptors are attached to receptor specific antibodies or ligands for the receptors
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35
Q

BiFC

A
  • 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
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36
Q

Functions of accessory proteins

A
  • expression
  • binding
  • signalling
  • regulation
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37
Q

MRAP characteristics

A
  • Has 2 splice forms MRAPa and MRAPB
  • Single TM proteins
  • form anti-parallel homodimers
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38
Q

MC2R

A
  • poorly expressed at cell surface in noradrenal cells
  • mutations cause FGD
  • MRAP has association with FGD
39
Q

MRAP2 characteristics

A
  • orthologue of MRAP
  • single TM
  • also a dimer
40
Q

MC2R and MRAP

A
  • 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
Q

MC1R/MC3R and MRAP

A
  • 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
Q

MC4R/MC5R and MRAP

A
  • receptors are normally expressed at cell surface
  • when co-expressed with MRAP then transport from ER is blocked = decreased expression and signalling
43
Q

RAMP characteristics

A
  • family of 3 proteins
  • Single TM
  • long extracellular N terminus
  • Short intracellular C terminus
44
Q

CGRP receptor

A
  • 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
Q

CLR and RAMP1 cell surface expression

A
  • not expressed when alone
  • RAMP1 has an ER retention motif
  • when co-expressed = expression at cell surface = increased cAMP signalling
46
Q

RAMPs altering ligand specificity of CLR

A
  • 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
Q

Gepant migraine drugs

A

work by blocking CGRP binding via binding of both CLR and RAMP1

48
Q

RAMPS affecting trafficking of CLR from cell surface

A
  • RAMP2 = targets CLR for internalisation and degradation
  • RAMP3 = recycling endosome
49
Q

VPAC1 and RAMP2

A

increased IP signalling in a ligand dependent manner

50
Q

G protein a subunit structure

A
  • GTPase domain
  • helical domain which interacts with B subunit
51
Q

Conformational change in GPCR allowing for G protein interaction

A
  • 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
Q

Role of RGS

A

regulate G protein activity by interacting with the a subunit to promote the transition state for GTP hydrolysis = promotes inactive form

53
Q

B-arrestin characteristics

A
  • 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
Q

B-arrestin interactions with GPCRs - B-arrestin conformational change

A
  • 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
Q

B-arrestin interactions with GPCRs - GPCR conformational change

A
  • changes similar to that of G protein interaction
  • TM6 moves less ~10A
56
Q

B-arrestin shape shifting

A
  • different phosphorylation can cause different conformational change
  • different protein recruited = different response
57
Q

B-arrestin as a scaffold for signalling

A
  • 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
Q

What type of proteins are GIP

A

intracellular proteins

59
Q

Biased receptor

A
  • receptor biased for a specific signalling protein
  • receptor activated by a ligand is in a conformation specific to a signalling protei
60
Q

Biased ligand

A

each ligand generates a different conformation activating a different pathway

61
Q

Intracellular protein bias

A

intracellular proteins can affect receptor conformation thus altering which ligand can bind

62
Q

Cell type or system bias

A
  • not strictly bias
  • proteins expressed can be exclusive to cell type so activate different pathways thus appearing biased
63
Q

Biased agonist PAC1 receptor

A
  • 2 peptides = PACAP38 and PACAP27
  • PACAP38 activates both cAMP and pERK1/2
  • PACAP activates cAMP but not pERK1/2 = BIASED
64
Q

Biased agonists - beta blockers

A

some beta blockers antagonise cAMP signalling but activate pERK1/2

65
Q

Why is bias an important consideration for drug safety and screening

A

allows for detection of more drugs and potential side effects

66
Q

Measurement of arrestin recruitment/activation for screening

A

done via either energy transfer or a complementation approach

67
Q

Label free technology - drug screening

A
  • 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
Q

Types of GIPs that regulate GPCR function

A
  • G proteins
  • Arrestins
  • Signalling kinases = JAK2
  • scaffold proteins = NHERF
69
Q

3 roles of GIPs

A
  • GPCRs can signal directly via GIPS
  • can enhance G protein signalling
  • can promote recycling or degradation of GPCRs after endocytosis
70
Q

GPCRs can signal directly via GIPS - Angiotensin I receptor

A
  • 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
Q

GIPs enhance G protein signalling - PTHI receptor

A
  • some cells preferentially couples to GaS = cAMP signalling
  • some cells express NHERF which causes Gaq signalling due to recruitment of downstream proteins
72
Q

GIPs promote degradation of GPCRs - u-opiod receptor

A
  • normally recycled after interaction with B-arrestin
  • when GASP1 is expressed the receptor is targeted for lysosomal degradation
73
Q

GIPs promote recycling of GPCRs - B2 adrenergic receptor

A
  • normally receptor is degraded after internalisation
  • when NHERF is present then it is recycled
74
Q

Methods for tracking GPCRs

A
  • 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
Q

Epiptope tags

A
  • 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
Q

Marker examples

A
  • EEA1 antibody = early endosome
  • calnexin = ER
  • DAPI dye = nucleus
77
Q

2 types of lipid raft microdomains

A
  • planar lipid raft
  • caveolae
78
Q

Planar lipid raft characteristics

A
  • continuous with plane of the membrane
  • promotes clustering of proteins
79
Q

Caveolae characteristics

A
  • create invaginations in membrane
  • 25-100nm diameter
  • cholesterol and sphingomyelin rich assemblies
  • contain caveolin protein
  • meeting point for cell signalling proteins
80
Q

Caveolae mechanisms

A
  • 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
Q

Scaffolds (lipid raft microdomains) changing specificity and activity

A
  • can change the function of a protein that bound by causing a conformational change
  • cytoskeletal proteins may help scaffold and organise
82
Q

B-arrestin mediated endosomal signalling

A
  • 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
Q

Adenylate cyclase mediated endosomal recycling - TSH receptor

A
  • 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
Q

Signalling in breast cancer - CXCR4

A
  • 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
Q

Viruses in cancer - KSAV

A
  • 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
Q

GPCR mutation causing cancer - TSHR

A
  • associated with thyroid cancer
  • cluster of mutations on or around TM6
  • mutations disrupt interactions between TM3 and TM6 = constitutively active
87
Q

Receptors involved in insulin release and secretion

A
  • 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
Q

Integration of GPCRs and glucose transporters in insulin release regulation

A
  • 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
Q

Diabetes drugs

A
  • K+ channel blockers
  • GLP-1 mimetics
  • work to increase GaS signalling = stimulate insulin release
90
Q

B-arrestin2 KO mice models - morphine and opioid

A
  • 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
Q

Arrestin knock in mouse models

A
  • 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
Q

Receptor bias

A

receptor has conformation that is bias for one signalling protein thus causing a specific signalling pathway

93
Q

Ligand bias

A

different ligands binding to the same receptor cause different signalling pathways due to different conformations

94
Q

Intracellular protein bias

A

intracellular protein can influence which ligand is bound to the receptor

Biosci 353 (2 decks)

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