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Cell signalling > GPCRs after 17 > Flashcards

GPCRs after 17 Flashcards

1
Q

3 methods of down regulation

A

Nucleotide exchange
Desensitisation- PKA/PKC or GrkS
Subunit swapping

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2
Q

Desensitisation of BAR

A

PKA acts on c3 and tail
7 BARK sites on tail (Ser/thr)
Reversed by phosphodiesterase

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3
Q

Heterologous desensitisation

A

GPCR -> PKA
Non specific of whether receptor is activated
Activation of one can desensitise another

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4
Q

Homologous desensitisation

A

GRK-1- rhodopsin kinase
GRK-2- BARK

GRK2
Recruited by surplus GBY subunits (isoprenylated) 
GRK-2 has a PH domain that binds BY
B-arrest in docks
Endocytosis
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5
Q

Functions of BY

5

A 
Inactive complex
Anchoring via isoprenylation
MACH -> BY acts on K+
Regulates AC 2,4
Regulation of BARK
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6
Q

b arrest in complex

A

Clathrin and AP2 -> endocytosis

AJK-1, MKKY, JNK-1 -> c-Jun cascade

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7
Q

B arrestin scaffold and dynamin

A

Interaction of B arrestin with c-src
Causes internalisation as promotes Yp of dynamin
C-src and dynamin mutants may inhibit clathrin dependent internalisation

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8
Q

Clinical application of receptor internalisation

A 
CCR5 to prevent R5 HIV-1 entry
Glycoprotein 120 can't bind CCR5
Uses agonists or antagonists 
Only affects CCR5 not CXC4 
Without inducing chemotaxis in vitro
Agonist ESN-196
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9
Q

3 pathways of GRK2/BARK activity

A

Recruits PKA, PKC, SRC, ERK AND CAM

Sequesters Gaq to prevent coupling to PLC

B arrestin recruitment. Allows PDE4 to membrane, degrades high camp levels

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10
Q

Two different PDEs recruited by B1 and B1 ARs in myocytes

A

B1- preformed with PDE48. PKA causes PDE dissociation

B2- recruitment of arrestin, which then recruits PDE4D5

Means that the different splice variants of PDE can cause contraction (B1) or inhibition (B2)

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11
Q

AKAP

A

Camp dependent protein kinase anchoring protein

Can localise PKA, PDE etc.

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12
Q

Camp pathway for lung disease treatment

A 
B2 agonist (albuterol)
AC agonist, forskolin
M2 antagonists (ipratropiumbromide)
PDE inhibitors, specific for Pde4
All act to increase camp
MLCK-Pi to increase bronchodilation
Phosphorylates K channel to stop leaving
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13
Q

Role of mAKAP in heart muscle

AKAP15

A

Anchors PKA and PDE
To nuclear membrane
Local regulation of camp levels
PKA can act on PDE which activates and lowers camp

AKAP15- cytosolic side near Ca channel.
PKA activates Ca
The AKAP15 localises PKA to the Ca channels

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14
Q

AKAP complexes

A 
B2AR
AC
PKA
EPAC (acts as a GEF for rap1)
PDE
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15
Q

Rhodopsin basic

A

Opsin (GPCR) linked to 11-cis-retinal
Transduction is the G protein
PTX (R174) and CTX (C347) sensitive

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16
Q

Rhodopsin and light transaction

A

Light causes photo isomerisation of 11-cis retinal -> all trans retinal
Forms unstable meta-rhodopsin II
This activates Gt
Gt acts to removes inhibitory Y of cGMP PDE
cGMP -> GMP
cGMP gated NA/ca close
Hyperpolarisation stops neurotransmitter -> relay to brain
Gt/PDE complex recruits RGS9-GB5
GTPase activity
PDE inactivated

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17
Q

Switching off the rhodopsin cascade

A

Low Ca activates guanylyl cyclase
Increased cGMP re opens gate
Ca entry and end to hyperpolarisation
Also GAP

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18
Q

Inhibition of the cascade by GRK1

A

Only light activated rhodopsin
Binds to C3 loop
Arrestin binds to phosphorylated opsin C-terminus
Always membrane associated
Recoverin- inhibits rhodopsin at high Ca (no light)
This stops GRK from being always active, so only really active st very low Ca/very bright light

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19
Q

Activation of gene expression by GOCRs

A

Increased camp
PKA catalytic units trans located to nucleus
Phosphorylate S133 of CREB
CREB binds to CRE
Associates with co factor CBP/P300 which is a histone acetylase
Transcription

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20
Q

PI -> DAG and ip3

A

PI -> PIP with PI-4 kinase
PIP -> PIP2 with PIP-5 kinase
PLC (cuts phosphoester bond)
G protein can act on PLC- Gq, Gao and Gat

Gives PKC activation

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21
Q

Why is Gq PTX resistant?

A

Lacks a Cys in the c terminal hexapeptide stretch

No ribosylstion target

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22
Q

Receptors coupled to PIP2 hydrolysis

A 
Muscarinic
H1
P2
A1
Thrombin
Vasopressin
Serotonin (only 5HT2)
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23
Q

Serotonin receptor subtypes

Types 1-7

A 
1- Gi camp
2- Gq -> IP3 DAG
4- Gs camp
5- Gi
6+ 7 Gs
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24
Q

Isoforms of PLC

A

Beta- activation by GPCR, Gaq and Gao

Gamma- activation by RTK, activated by phosphorylation

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25
Q

Bifurcating PI pathway

A

Write it out

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26
Q

Domain structure of PKC
How is it activated
Phorbal esters

A

Pseudo phosphorylation site near the N terminus
Inert alanine instead of Ser/thr
Binding of ca, DAG and phosphatidylserine activate (localises to membrane)
Binds to DAG and phorbal esters through zinc fingers

TPA (tetra phorbal acetate) and PMA (phorbal myristate acetate)
Act as DAG analogues

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27
Q

PKA and PKC comparison

A

BAR -> PKA -> TRE by Jun and Fos

A1AR -> PKC -> CRE by CREB

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28
Q

Fates of DAG

A

DAG lipase creates AA

Cyclooxygenase converts this to Prostaglandins and Thromboxanes

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29
Q

Fate of IP3

A

Release of Ca from ER
Back to inositol
Or used to create Ip4, ip5 or ip6

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30
Q

Biphasic release of Ca
Two phases
Why is one short lived?

A 
Phase I
Fast spikes
IP3 receptor of calciosomes
Action is specific, saturable and reversible
Due to Ca ATPases
And quick hydrolysis of IP3 to IP2

Phase II
Long lived
IP4 opens the Ca Chanel at the cell membrane

31
Q

Calcium acting as a co agonist

A

Ip3R not just controlled by IP3
Ca acts as a coagonist
Low Ca encourages IP3R opening
Max Ca concentration of 2-3 mM

32
Q

Direct and indirect methods to measure calcium

A

Direct
Fluorescent probes- Bind free Ca and change characteristics
Photo protein aequorin emits light on Ca binding
ATP added to thymus cells to raise Ca, or hepatocyte and adrenaline

Indirect
Ca electrodes to measure flow of Ca across membranes

33
Q

Ca spikes in pituitary glands

A

Cells secreting LH
Induced by binding of LHRH to its GPCR
Repeated ca spikes
Each spike causes exocytosis of a few LH vesicles

34
Q

Regulation of Ca spikes

STIM1

A

Oscillations. Conversion of conc -> frequency
PLC and Ion channels regulate levels
STIM1 in the ER lumen can bind to the Ca channel of the PM
Draws the two membranes together when there is low ER Ca

35
Q

Levels of calcium

A

Steady state 10-7
Extra cellular 2 mM
Maintain a calcium gradient

36
Q

What cell functions does Ca control?

A 
PKC
kinases e.g. PKC 
PDE and AC
Metabolic enzymes
Liver to activate GPK via PKC
Nitric oxide synthase signalling
37
Q

Negative feedback and cross talk in heart muscle cells

2 GPCR pathways

A

Adrenaline
B1 -> camp -> PKA -> PDE3 to lower camp

A1 -> Gq to activate PLC -> Ip3 -> Ca increase -> PDE1
Decreases camp produced by the B1

38
Q

Contraction regulation of smooth muscle by NO and cGMP

A 
ACH -> GPCR -> PLC -> IP3 -> Ca 
Activation of NO synthase
Converts Arg + O2 -> Citrulline + NO
Binds to No receptor of muscle
Converts GTP -> cGMP
cGMP activates PKG -> myosin LC -> relaxation
39
Q

Cell responses of different tissues to Ca increase

A

Pancreas -> digestive enzymes

Smooth muscle -> contraction

40
Q

How do we turn off Ca signalling?

A 
Nucleotide exchange
Desensitisation
RGSs
Regulation of secondary messengers
GRK2 can bind to Gaq
41
Q

Selective regulation of Gq by GRK2

A

Kinase dead GRK2 still decreased IP production
Phos independent mechanism
Specifically n terminus of grk2
Grk2 has an RGS domain

42
Q

Different hormone classes

A

Peptide- insulin, glucagon -> GPCRs and RTKs

Steroid- testosterone -> nuclear receptors

43
Q

Hormones which use nuclear receptors

A

Steroid- glucocorticoids (cortisol), gonadal steroids

Vit D

Thyroid hormones- thyroxine

Retinoids hormones- retinol (Vit A precursor)

44
Q

Vitamin D

A

7-dehydrocholesterol
Photolysis
Vit D3 (cholecalciferol)
Hydroxylstion to 25-hydroxycholecalciferol
1a,25-dihydroxycholecalciferol (calcitrol)

Then acts through NRs to activate Ca binding protein for Ca uptake in intestines

45
Q

Nuclear receptor examples

A

ER, AR PR- proliferative
GR- anti proliferative
25 orphans

46
Q

PEPCK

A

Converts oxaloacetate to PEP

After pyruvate -> OA by pyruvate carboxylase

47
Q

Activation of genes by GR

A 
Homodimeric
Hormone removes Hsp90 
Dimerisation
Translocation
DBD binds to GRE so that the activation domain at the N terminus regulates transcription
48
Q

Sedimentation coefficients

A

Monomeric 3-5s
Dimer is 6-10s
More salt and temp favours activation
Causes AF2 unfolding to bind coactivators

49
Q

N terminal hyper variable activation domain

A

50-500 most variable length and sequence
AF-1 region important for receptor dependent trans activation- binds the transcription machinery
Co regulators can bridge af1 and 2 to make ligand independent
More folded af1 when protein protein interactions
Ser/thr sites for regulation
Allows priming for trans location

50
Q

DNA binding domain

A

Central, 68 AAs
Conserved
2 copies of C4 zinc finger, 2 per monomer
8 Cys that coordinate Zn. 2 helices folded perpendicular
Hydrophilic and basic
Interacts with PO4 backbone

51
Q

Variable hinge region

A

18 AAs

NLS

52
Q

C-terminal ligand binding domain

A 
200-250 AA
12 helices, 3 sets form ligand pocket
Hydrophobic AA for steroid interaction
R dimerisation by helices 9 and 10
Protected by hsp90
AF-2 on H12 for trans activation, LBD surface for coactivators and corepressors such as histone deacetylases
53
Q

Carboxyl F domain

A

No known function

54
Q

Roles of chaperones on GR signalling

A

Regulates folding
Prevents nuclear entry
Removal of hsp90 by salt causes LBD collapse
Hsp binds to hydrophobic
Changes protein to open cleft for ligand binding
No DNA binding when in complex
Protects from ubiquitin ligase

55
Q

Conservation of NR domains

A

Variable 0
DNA 42-94%
Ligand 15-57%

56
Q

3 families of nuclear receptors

A

GR
ER
non steroid

57
Q

Chimeric receptor proteins NRs

Experiment to prove modular

A

Can swap the DBD and LBD
Swapped GR and retinoic acid

Fused b-galactosidase to different parts of GR then blotted with fluorescent antibody
Used dexamethasone which is synthetic Glucocorticoid
Shows LBD needed for translocation

58
Q

Zinc finger motif

A

Monomers have 2x zinc finger
Zn surrounded by 4 Cys
Inserts in to major groove of DNA of HRE

59
Q

Type I homodimeric NR

A 
AR, PR, GR
Cytoplasm with Hsp
Homodimers, inverted DNB
Binding of activated R to positive HRE
Trans activation, chromatin remodelling
Binding to negative would give repression
60
Q

Type II heteromeric NR

A

VDR, RAR, TR, RXR
Located in nucleus
Always associated with HRE
Heterodimers with RXR (9-cis retinoic acid)
Repress transcription, associated with histone deacetylases
Silences promoter

Activation destabilises the corepressor binding site of LBD
Gives hyper acetylation of histones

61
Q

Degradation of NRs

A

Ubiquitin protea some pathway

Targets for phosphorylation so can cross talk

62
Q

4 ways that GR can control gene expression

A

Transactivation
Transrepression
Fos/Jun - bind to AP1 regulatory site. Effect reduced with GR
Nuclear factor kB- transcription factors P65/50 bind to the NFkB site
And promote gene transcription, prevented by GR

63
Q

Common features of HREs

A

Upstream
15-20, dead symmetry
Basic consensus, but with slight variation

64
Q

Which have palindromic and direct repeats?

Significance of spacing?

A

GRE ERE - 6bp inverted. N=3
VDRE TRE RARE- direct repeats. N 1-5

Spacing affects the position of the second nuclear receptor
Addition of 1 BP gives a 35 degree rotation and 3.4A separation

65
Q

3 key features of HREs

A

Spacing
Orientation
Sequence

66
Q

Interaction of HREs in the lysozyme gene and casein

A

PRE and GRE overlap
Synergistic

In casein, the GRE stops transcription

67
Q

Experiment to determine which section of DNA the NR binds to

A

Use DNAse
DNA protected by protein is the region that it binds
Or site directed mutagenesis

68
Q

How do zinc fingers distinguish between HREs?

A

E25 G26 A29 - PBOX, recognise ERE

P44-Q48 - DBOX, for spacing. How big palindrome is (homo) and space between the tandem half sites (hetero)

69
Q

Finger swapping experiments

A

Change the p box to make recognise a different response element
By using point mutations increase the affinity for GRE instead of ERE etc.
EG–A -> GS–V

70
Q

Clinical implications
Mutation in Vit d3 receptor
Mutation in androgen receptor

A

Inactive VDR, lack of Vit d3

XY syndrome, androgen insensitivity syndrome

71
Q

Tamoxifen

A

Treatment of oestrogen dependent breast cancer
Competitive ligand
Doesn’t promote gene expression so slows cancer

Locks in inactive confirmation
Helix moves to sterically block coactivators
Helix 12 from LBD can’t interact

72
Q

RU 486

A

Progesterone antagonist

Stops implantation

73
Q

Glucocorticoid treatment

A

Dexamethasone

Reduces inflammatory gene transcription

Cell signalling (5 decks)

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