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Cell signalling > GPCRs after 17 > Flashcards

GPCRs after 17 Flashcards

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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
Bifurcating PI pathway
Write it out
26
Domain structure of PKC How is it activated Phorbal esters
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
27
PKA and PKC comparison
BAR -> PKA -> TRE by Jun and Fos | A1AR -> PKC -> CRE by CREB
28
Fates of DAG
DAG lipase creates AA | Cyclooxygenase converts this to Prostaglandins and Thromboxanes
29
Fate of IP3
Release of Ca from ER Back to inositol Or used to create Ip4, ip5 or ip6
30
Biphasic release of Ca Two phases Why is one short lived?
``` 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
Calcium acting as a co agonist
Ip3R not just controlled by IP3 Ca acts as a coagonist Low Ca encourages IP3R opening Max Ca concentration of 2-3 mM
32
Direct and indirect methods to measure calcium
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
Ca spikes in pituitary glands
Cells secreting LH Induced by binding of LHRH to its GPCR Repeated ca spikes Each spike causes exocytosis of a few LH vesicles
34
Regulation of Ca spikes | STIM1
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
Levels of calcium
Steady state 10-7 Extra cellular 2 mM Maintain a calcium gradient
36
What cell functions does Ca control?
``` PKC kinases e.g. PKC PDE and AC Metabolic enzymes Liver to activate GPK via PKC Nitric oxide synthase signalling ```
37
Negative feedback and cross talk in heart muscle cells | 2 GPCR pathways
Adrenaline B1 -> camp -> PKA -> PDE3 to lower camp A1 -> Gq to activate PLC -> Ip3 -> Ca increase -> PDE1 Decreases camp produced by the B1
38
Contraction regulation of smooth muscle by NO and cGMP
``` 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
Cell responses of different tissues to Ca increase
Pancreas -> digestive enzymes | Smooth muscle -> contraction
40
How do we turn off Ca signalling?
``` Nucleotide exchange Desensitisation RGSs Regulation of secondary messengers GRK2 can bind to Gaq ```
41
Selective regulation of Gq by GRK2
Kinase dead GRK2 still decreased IP production Phos independent mechanism Specifically n terminus of grk2 Grk2 has an RGS domain
42
Different hormone classes
Peptide- insulin, glucagon -> GPCRs and RTKs | Steroid- testosterone -> nuclear receptors
43
Hormones which use nuclear receptors
Steroid- glucocorticoids (cortisol), gonadal steroids Vit D Thyroid hormones- thyroxine Retinoids hormones- retinol (Vit A precursor)
44
Vitamin D
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
Nuclear receptor examples
ER, AR PR- proliferative GR- anti proliferative 25 orphans
46
PEPCK
Converts oxaloacetate to PEP | After pyruvate -> OA by pyruvate carboxylase
47
Activation of genes by GR
``` Homodimeric Hormone removes Hsp90 Dimerisation Translocation DBD binds to GRE so that the activation domain at the N terminus regulates transcription ```
48
Sedimentation coefficients
Monomeric 3-5s Dimer is 6-10s More salt and temp favours activation Causes AF2 unfolding to bind coactivators
49
N terminal hyper variable activation domain
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
DNA binding domain
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
Variable hinge region
18 AAs | NLS
52
C-terminal ligand binding domain
``` 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
Carboxyl F domain
No known function
54
Roles of chaperones on GR signalling
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
Conservation of NR domains
Variable 0 DNA 42-94% Ligand 15-57%
56
3 families of nuclear receptors
GR ER non steroid
57
Chimeric receptor proteins NRs | Experiment to prove modular
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
Zinc finger motif
Monomers have 2x zinc finger Zn surrounded by 4 Cys Inserts in to major groove of DNA of HRE
59
Type I homodimeric NR
``` 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
Type II heteromeric NR
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
Degradation of NRs
Ubiquitin protea some pathway | Targets for phosphorylation so can cross talk
62
4 ways that GR can control gene expression
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
Common features of HREs
Upstream 15-20, dead symmetry Basic consensus, but with slight variation
64
Which have palindromic and direct repeats? | Significance of spacing?
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
3 key features of HREs
Spacing Orientation Sequence
66
Interaction of HREs in the lysozyme gene and casein
PRE and GRE overlap Synergistic In casein, the GRE stops transcription
67
Experiment to determine which section of DNA the NR binds to
Use DNAse DNA protected by protein is the region that it binds Or site directed mutagenesis
68
How do zinc fingers distinguish between HREs?
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
Finger swapping experiments
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
Clinical implications Mutation in Vit d3 receptor Mutation in androgen receptor
Inactive VDR, lack of Vit d3 XY syndrome, androgen insensitivity syndrome
71
Tamoxifen
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
RU 486
Progesterone antagonist | Stops implantation
73
Glucocorticoid treatment
Dexamethasone | Reduces inflammatory gene transcription

Cell signalling (5 decks)

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