Signal termination of G protein coupled receptors 2018 english… · Signaltermination of G protein...

Signal termination

of G protein coupled receptors

Cell

SIGNAL:

Hormons

Neuro transmitters

Cytokines

Light

Odorant

Prostaglandins

REACTION:

Change in metabolism

Protein synthesis

mRNA

Differentiaton

Morphologiy

Signal transmisson

Function of signal termination

. Protection for permanent cell stimulation

. Adaption of the cellular response

. Integration of signal chains

Short term signal termination - short term signal -

(sec-min)

fast adaption to signal changes

Reversible

Inactivation of signal molecules

(by chemical modification,

cellular redistribution of signal molecules,

protein/protein interaction)

Long term signal termination - long term signal –

(hours-days)

Irreversible


(by proteolytic degradation,

inhibition of de novo synthesis)

permanent adaption to chronical

signal changes

Short term signal termination - short term signal -

(sec-min)

Reversible


(by chemical modification,

cellular redistribution of signal molecules,

protein/protein interaction)

fast adaption to signal changes

Signaltermination of G protein coupled receptors

(GPCR)

- signal termination of this receptor system is very well characterised

- more than 2000 papers published in this field !

Reason:

- 100.000 genes in the human genom 1000 genes coding for GPCR!

- interesting target for the pharmaceutical industrie

- broad spectre of ligands: neurotransmitter,s mormons,

chemokines, prostanoids, light, odorants

- involved in severe disease: high pressure, chronisc heard failure,

inflammation, HIV, opiat dependence

Signal transduction of G protein coupled receptors

e

i

PM

circulating ligands

NH2-

- COOH

Receptor

Odorants, light

e

i

PM

NH2-

- COOH a b g

G-Protein Receptor

a

GDP

GTP b g


circulating ligands Odorants, light

e

i

PM

NH2-

- COOH a b g

G-Protein Receptor

Effectors (z. B. AC, PLC, ion chanels, MAPK)

a

GDP

GTP b g



i

PM

NH2-

- COOH a b g

G-Protein Receptor

Effectors (z. B. AC, PLC, ion chanels, MAPK)

second messenger (cAMP, IP3, DAG, Ca2+)

a

GDP

GTP b g



i

PM

NH2-

- COOH a b g

G-Protein Receptor

Effektors (z. B. AC, PLC, ion chanels, MAPK)


a

GDP

GTP b g

Protein kinases (PKA, PKC)



Decrease of hormone concentration

signal termination

e

i

PM

NH2-

- COOH

H [HR] = [H] [R]

Kd H

Signal

e

i

PM

NH2-

- COOH

H [HR] = [H] [R]

Kd H

Signal

external signal endocrine system transient [H]


signal termination

e

i

PM

NH2-

- COOH

H [HR] = [H] [R]

Kd H

Signal

[H] near Kd of the receptor signal


signal termination


e

i

PM

NH2-

- COOH

H [HR] = [H] [R]

Kd H

Signal


[H] near Kd of the receptors signal

[H] [HR] signal termination


signal termination

Decrease of agonist concentration

- Mechanismn -

1. Dilution

2. Uptake by specific transporters

3. Extracellular degradation

4. Receptor-mediated endocytosis

Active uptake of the agonist (synapsis)

cocaine Sarin

sarine


cocaine

sarine


cocaine

e

i

PM

NH2-

- COOH a b g

G-Protein Receptor

Effektors (z. B. AC, PLC, Ionenkanäle, MAPK)


a

GDP

GTP b g




Desensitisation of G protein coupled receptors

(GPCR)

• Homologous Desensitisation

- Exclusive inhibition of the active,

ligand-bound receptor

Signal termination of the receptor

• Heterologous Desensitisation

-Inhibition of a receptor after

the activation of a second,

heterologous receptor

Desensitisation of G protein coupled receptors

(GPCR)

Signal termination of the receptor

• Homologous Desensitisation

- Exclusive inhibition of the active,

ligand-bound receptor

Desensitisation und phosphorylation von GPCR

Example: Angiotensin II receptor:

Ligand: Angiotensin II Function: potenter regulator of blood pressure

G-Protein: Gq second-messenger: InsP3 , Ca2+

Rezeptordesensitisation:

Receptor phosphorylation:

Desensitisation und phosphorylation von GPCR

Example: Angiotensin II receptor:

Ligand: Angiotensin II Function: potenter regulator of blood pressure

G-Protein: Gq second-messenger: InsP3 , Ca2+

Rezeptordesensitisation:

G protein coupled receptor kinases (GRKs)

Receptor recognition

Membrantargeting

GRK features

Name Intracelluar localisation

Receptor substrate Tissue

GRK 1 Rhodopsin

Kinase Plasmamembrane Rhodopsin (ß2-AR) retina

GRK 2 ßARK 1 Cytosol ß2-AR, a2-AR, m2-ACh-R,

Thr-R, AT II-R etc.

leukocytes, heart, brain, lung, kidney

GRK 3 ßARK 2 Cytosol ß2-AR, a2-AR, m2-ACh-R,

Thr-R, AT II-R etc.

spleen, heart, brain, lung, kidney

GRK 4 - Plasmamembrane ß2-AR, LH-R testis, (brain) GRK 5 - Plasmamembrane ß2-AR, Rhodopsin muscle, heart, liver

brain, lung, kidney GRK 6 - Plasmamembrane ß2-AR, Rhodopsin muscle, heart, liver

brain, lung, kidney

Agonst-induced phosphorylation of GPCR by GRKs

inactive receptor active receptor

GRK-Phosphorylation - only agonist-bound receptor is substrat

- phosphorylation of serine and threonin residues

- no consensus sequence

- allosteric phosphorylation

- membrane targeting is essentiell















P

1 2 3 4 5 6 7

bg

GRK 2/3

GRK 5/6

S/TS/T

A

Regulation of GRK phosphorylation

acivated receptor

P

GRK 2/3

+

P

1 2 3 4 5 6 7

bg

GRK 2/3

GRK 5/6

S/TS/T

A


activated receptor

P

GRK 2/3

+

+

P

1 2 3 4 5 6 7

bg

GRK 2/3

GRK 5/6

S/TS/T

A


activated receptor

P

GRK 2/3

PIP2

+

+

+

+

P

1 2 3 4 5 6 7

bg

GRK 2/3

GRK 5/6

S/TS/T

A

Regulation of GRK phosphoryation

activated receptor

P

GRK 2/3

PIP2

+

+

PKC

+

+

+

+

Re


activated receptor

Heterologous receptor desensitisation and phosphorylation

Example: ß2-adrenergic- und M3-muscarinic receptor

Ligand: ß2-AR: Isoproterenol

M3-R: Carbachol

G-protein: ß2-AR: Gs, cAMP

M3-R: Gq, IP3, Ca2+




M3-R: Carbachol

heterologous receptor desensitisation


M3-R: Gq, IP3, Ca2+

1. Prä-Stimulus: Carbachol

2. Stimulus: Isoproterenol




M3-R: Carbachol

heterologous receptor desensitisation heterologous receptor phosphorylation


M3-R: Gq, IP3, Ca2+

1. Prä-Stimulus: Carbachol

2. Stimulus: Isoproterenol 1. Phosphorylierung of ß2-AR

by stimulation with carbachol

Phosphorylation of GPCR by

second-messenger dependent kinases

ß2-adrenergic receptor M2-muscarinic receptor

Phosphorylation of GPCR by

second-messenger dependent kinases

ß2-adrenergic receptor M2-muscarinic receptor

Interplay of GRKs and second-messenger dependent

kinases in GPCR desensitization

Example: Olfactoric receptors

Arrestin-induced uncoupling of receptor and G protein

activated receptor

AV

CP

L

L

LN

Q

L

P

W

VA

E

G

I

S

I

S

L

K

S

E

S

P

K

SR

Q

SEV

L

A

R

K

D

A

extracellular

intracellular

K

V

C

CL

FRIG GSRR

E

EQ QH

C

C

C

R

T

TS S SSS AMH

G G

GF

R

R

S S

S

S T

T

I

I

IE

L K E Q T L L P

D

D

D

LLLL S

S

S

S

P N G

GG

G

R

R

R

R N

L

l

L L PP

P V V

VM

A AA

A

Q

Q

Q

QE EE

EE

L

LL

T

T

SS S

S

S ST T

G

G GG D

D

LWK KGSS L LVFPNLE

N

T

R

K V

D

COOH

I

D P

V

L

l

l

l

L

l

l

L

Y

R

R

Q

CHO

335

484

SP

Y

D S

370382

hEP4-R wild type

EP4-R: Identiffication of serine/threonine residues

essentiell for EP4-R/ß-arrestin interaction

Phosphorylierungsstelle

AV

CP

L

L

LN

Q

L

P

W

VA

E

G

I

S

I

S

L

K

S

E

S

P

K

SR

Q

SEV

L

A

R

K

D

A

extracellular

intracellular

K

V

C

CL

FRIG GSRR

E

EQ QH

C

C

C

R

T

TS S SSS AMH

G G

GF

R

RS

A

T

I

I

IE

L K E Q T L L P

D

D

D

D LLLL S

S

S

S

P N G

GG

G

R

R

R

R N

L

l

L L PP

P V V

VM

A AA

A

Q

Q

Q

QE EE

EE

L

LL

T

T

SS S

S

S ST T

G

G GG D

D

LWK KGSS L LVFPNLE

N

T

R

K V

D

COOH

I

D P

V

L

l

l

l

L

l

l

L

Y

R

R

Q

CHO

335

484

SP

Y

A A A S

389-392

370382

hEP4-ST389-392A-R

Phosphorylierungsstelle

Arrestin-

interaction

EP4-R: Identiffication of serine/threonine residues

essentiell for EP4-R/ß-arrestin interaction

Arrestin: Adaptor in GPCR desensitisation

Phosphorylierungserkennung Clathrinbindung

Erkennung des akt. Rezeptors

48 kD

Arrestins:

Tissue

Localisation

Vis. Arrstin

Retina

Cytosol

ß-Arrestin 1 und 2

ubiquitious

Cytosol

cone-Arrestin

Retina, Lung

Cytosol

D- E-Arrestin

ubiquitious

Cytosol

Struktur

recognition of phosphorylation clathrin binding

recognition of the activated receptor

48 kD

Structure

Arrestin: Adaptor in GPCR desensitisation

Arrestins:

Tissue

Localisation

Vis. Arrstin

Retina

Cytosol

ß-Arrestin 1 und 2

ubiquitious

Cytosol

cone-Arrestin

Retina, Lung

Cytosol

D- E-Arrestin

ubiquitious

Cytosol

Agonist-induced sequestration von GPCR

Function of arrestin

Example: Adenosin receptor

G-Protein: Gs

Localisation of receptor and arrestin

receptor

Arrestin-GFP

Rezeptor

+

Arrestin-GFP

receptor

Arrestin-GFP

Rezeptor

+

Arrestin-GFP

Agonist-induced sequestration von GPCR

Function of arrestin

Example: Adenosin receptor

G-Protein: Gs

Localisation of receptor and arrestin

receptor

Arrestin-GFP

Rezeptor

+

Arrestin-GFP

receptor

Arrestin-GFP

Rezeptor

+

Arrestin-GFP

Sequestration of GPCR




Recycling and resensitization of GPCR

recycling resensitization

lysosomal degradation

of the receptor

(down regulation)




of the receptor

(down regulation)




of the receptor

(down regulation)

e

i

PM

NH2-

- COOH a b g

G-Protein Receptor



a

GDP

GTP b g




GTPase function

Signal termination of G proteins

Signal termination of G proteins

GTPase function

in-vitro extremely slow

SIGNALTERMINATION of G proteins

GTPase Activating Protein

Regulators of G protein Signalling



RGS proteins accelerate the change of

Ga-GTP to Ga-GDP







all RGS proteins act as GAPs!!


e

i

PM

NH2-

- COOH a b g

G-Protein Receptor



a

GDP

GTP b g




Inactivation of second-messengers

Phosphatase

(inhibited by Li)

inaktiv

inaktiv

Insositol

Inactivation of second-messenger

inaktiv

inaktiv

Insositol

Diacylclycerin

Phosphatidylinositol

Insositol

Inactivation of second-messengers

Phosphatase

(inhibited by Li)

e

i

PM

NH2-

- COOH a b g

G-Protein Receptor



a

GDP

GTP b g




Inactivation of protein kinase A (PKA)

by feedback-inhibition

SUMMARY

Signal termination acts on all steps of the signal chain

1. Agonist:

- Dilution, uptake, degradation

2. Receptor:

- Phosphorylation, uncoupling of the G protein,

internalisation, recycling, degradation, inhibition of synthesis

3. G-Protein:

GTPase function, GAPs and RGS-proteins

4. second-Messenger:

Hydrolysis, dephosphorylation, reesterication

5. Effector enzymes (kinases):

Feedback inhibition

SUMMARY

Signal termination acts on all steps of the signal chain

Date post:	15-Jul-2020
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Signal termination of G protein coupled receptors 2018 english… · Signaltermination of G protein...

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