Transcript (2) G Protein-Coupled Receptors

Chapter 15
Cellular Signal Transduction
The biochemistry and molecular
biology department of CMU
When environment changes:
Monad——responds directly.
Multicellular organisms——signal
through elaborate system of
intercellular or intracellular
communication，and consequently
regulate functions of organisms.
Signaling molecule
Receptor of target cell
Signal
transduction
Intracellular molecule
biological effect
§1 Signaling Molecules
Signaling molecules
• Signaling molecules, which are
released by signal-producing cells,
reach and transfer biological signals
to their target cells to initiate specific
cellular responses.
• Extracellular molecules
• Intracellular molecules
1. Extracellular molecules
protein & peptides: Hormone, cytokine
AA & its derivatives: Gly, Glu, adrenaline,
thyroxine
Steroid: Sex Hormone,
glucocorticosteroid
Fatty acid derivatives: prostaglandin
(1) Paracrine signaling
(local chemical mediators)
• Secreted by common cells.
• Reach neighboring target cells by
passive diffusion.
• Time of action is short.
• Such as GF, PG
(2) Endocrine signal
•
•
•
•
Secreted by endocrine cells.
Reach target cells by blood circulation.
Time of action is long.
Such as insulin, thyroxine, adrenalin
(3) Synaptic signal
(neurotransmitters)
• Secreted by neuronal cells.
• Reach another neuron by synaptic
gap.
• Time of action is short.
• Such as Acetylcholine (Ach),
noradrenaline
(4) Gaseous signal
• Simple structure, half life is short
and active in chemistry .
• Such as NO, CO.
GAS MOLECULE
(5) Autocrine signal
• Act back to their own cells.
• Such as GF, cytokine, interferon,
interleukin.
2. Intracellular molecule
• Ca2+
ions
• DG, ceramide
• IP3
lipid derivatives
carbohydrate
derivatives
• cAMP cGMP
nucleotides
• Ras, JAK, Raf
proteins
Second messenger:
Small molecules synthesized in cells
in response to an external signal are the
second messengers, which are
responsible for intracellular signal
transduction.
Such as Ca2+, DG, Cer, IP3, cAMP,
cGMP
Third messengers:
Third messengers are the molecules
which transmit message from outside
to inside of nucleous or from inside to
outside of nucleous, also called DNA
binding protein.
Proteins and peptides:
Hormones, cytokines
Effect by
membrane Amino acid derivatives:
receptors
Catecholamines
Fatty acid derivatives:
Extracellular
molecules
Prostaglandins
Effect by
intracellular
receptors
Signal
molecules
Intracellular
molecules
Steroid hormones,
Thyroxine, VD3
cAMP, cGMP, IP3, DG, Ca2+
§2 Receptor
Receptor
Receptors are specific membrane
proteins, which are able to recognize
and bind to corresponding ligand
molecules, become activated, and
transduce signal to next signaling
molecules.
Glycoprotein or Lipoprotein
ligand
A small molecule that binds
specifically to a larger one; for
example, a hormone is the ligand for
its specific protein receptor.
• Membrane receptors
membrane
Glycoprotein
• Intracellular receptors
Cytosol or nuclei
DNA binding protein
1. membrane receptors
(1) Ligand-gate ion channels type
(cyclic receptor)
ligand→receptor→ion channel open or
close
(2) G Protein-Coupled Receptors
(serpentine R)
1) 7-helices transmembrane
receptor
Oligosaccharide
unit
Cytosolic
side
2) G protein (Guanylate binding
protein)
• G protein refers to any protein which
binds to GDP or GTP and act as signal
transduction.
• G proteins consist of three different
subunits (, , -subunit).
• -subunit carries GTPase activity,
binding and hydrolysis of GTP.
3) Classes of G protein
Gs→ s→AC→cAMP↑
Gi→ i→AC→cAMP↓
Gq→ q →PI-PLC→IP3+DAG
Go→ o→ion channel
Gt→ t →cGMP PDE→cGMP→
Rhodopsin
Glucagon
-adrenaline →s →AC↑
ACTH
-adrenaline
angiotensin Ⅱ
acetylcholine(M2 M4)
GF release inhibitory factor
→i→AC↓
Cholera toxin
Ribosylation of Arg of G
G s -ATP
ATPase
Gs -ADP
AC
cAMP
Cl- + H2O
HCO3-
Cavity of intestine
diarrhea
Pertussis toxin
i -ADP-ribosylation
Gi
AC↑
cAMP↑
allergy of histamine
4) Effect proteins of G protein
AC
cGMP PDE (phosphodiesterase）
PLC
PLA2
Channel protein
cAMP
ATP
5) Pathway of G protein linked
receptor
H
R
G protein
Es
secondary messeger
Protein kinase
Phophorylation of Es or functional protein
Biological effect
(3) Single transmembrane α-helix
receptor
• Tyrosine protein kinase Receptor
(catalytic receptor)
IGF－R, EGF－R
• Non tyrosine protein kinase Receptor
Growth Hormone R, interferon R
Tyrosine protein kinase Receptor
or receptor tyrosine kinase (RTK)
Cys-rich
domain
Immunoglobulin
-like
domain
EGFR
IGF-1R
PDGFR
FGFR
Insulin
Cytosol
Intracellular
insulin effects
Non-receptor tyrosine kinase
(NRTK)
Domain of Downstream molecules
of TPKR
SH2 domain (Scr homology 2 domain)
SH3 domain
PH domain (pleckstrin homology domain)
(4) Guanylate cyclase (GC)
receptor
Membrane receptor –ANP
Soluble receptor – NO, CO
2. Intracellular receptor
(transcription regulated receptor)
Intracellular R is trans-acting
elememt
cis-acting element
gene expression
Localized in the cytosol and/or in
the nucleus.
ligand: Steroid H, VD3, Thyroxine
3. Properties of binding of H
and R
• highly specificity
• highly affinity
• saturation
• reversible binding
• special function model
4. Control of receptor activity
• Phosphorylation or
dephosphorylation of R
• Phospholipid of membrane
• Enzyme catalyzed hydrolysis
• G protein regulation
5. Function of receptor
(1) Recognize the special ligand
(2) Binding to special ligand
(3) Signal transduction
biological effect
§3 Pathway of Signal
Transduction
Signal transduction mediated
by membrane receptor
• cAMP dependent-protein kinase A
pathway
• cGMP dependent PKG pathway
• Ca2+ dependent PK pathway
• Tyrosine protein Kinase pathway
• NF-κB pathway
• TGF- βpathway
1. cAMP dependent-protein
kinase A pathway
H
R
G protein
AC
cAMP
PKA
Phosphorylation of Es or functional proteins
Biological effects
(1) cAMP metabolism
ATP
AC
2+
PDE
cAMP
2+
Mg
Mg
PPi
H2O
PDE： Phosphodiesterase
AC： Adenylate cyclase
5'-AMP
NH2
N
N
O
O
P
O
O
O
P
O
O
P
O
CH2 O
ATP
H
H
O
O
N
N
H
H
OH
OH
AC
PPi
NH2
NH2
N
N
N
N
O
cAMP
H
H
O
P
O
O
H
H 2O
H
O
O
PDE
CH2 O
OH
N
N
P
N
N
O
5'-AMP
CH2 O
H
O
H
H
H
OH
OH
(2) Mechanism of cAMP effect
Activate cAMP-dependent
protein kinase (PKA).
(3) PKA effect
Phosphorylate specifically
Ser/Thr residues in several
proteins
（1）Regulation of
metabolism
（2）Regulation of gene
expression
hormons: glucagon, epinephrine
inactive AC
active AC
ATP
cAMP
ATP
inactive PKA
phosphorylase b kinase
Pi
active PKA
P
ADP
ATP
phosphorylase b kinase
ADP
ATP
ADP
P
glycogen
synthase
(active)
P
glycogen
synthase
(inactive)
phosphorylase b phosphorylase a
Pi
Pi
inhibitor-1
(inactive)
H2O
ATP
H2 O
protein
phosphatase-1
inhibitor-1
(active)
P
H2O
CRE : cAMP response element
(TGACGTCA)
CREB: CRE binding protein
CRE
DNA
Transcription
mRNA
CREB
PKA
Ｐ
CREB
PKA
Ｐ
2. Ca2+ dependent PK pathway
(1) Ca2+ -DAG -dependent PKC pathway
H
R
PIP2
PLC
G protein
IP3
ER
DG
Ca2+ PS
PKC
Phosphorylation of Es or functional proteins
Biological effects
[Ca 2+]i 0.01-1 mol/L（10-7 mol/L ）
[Ca 2+]o 2.5mmol/L（10-3 mol/L ）
5000~10000×
1） Function of DG and IP3
IP3 + R→open of Ca2＋
channel →[Ca2＋]↑
DG
PS, Ca2＋
PKC ↑
2）Function of PKC
• regulation of metabolism
PKC →Ser/Thr-P of R, enzyme,
Protein of Mb.
• Gene expression
Early response:
PKC
Trans-acting factor-P
Immediate early genes
Third messenger
Late response:
PKC
Third messenger-P
Activate genes
Cell proliferation
(2) Ca2＋-CaM dependent protein
kinase pathway
H
R
G protein
PLC
IP3
Ca2+
CaM
CaMK
Phosphorylation of Es or functional proteins
Biological effects
Calmodulin (CaM）：
Ca2＋ binding protein
4 Ca2＋+ CaM → Ca2＋- CaM
↓
CaM kinase↑
↓
Ser/Thr - P
↓
Ca2＋ pump, AC ↑ GC ↑
Es (glycogen synthase, phosphorylase
kinase)
3. cGMP-dependent PKG pathway
ANP
NO, CO
Receptorlinked GC
cGMP
Soluble GC
PKG
Phosphorylation of Es or functional proteins
Biological effects
(1) cGMP
GTP
GC
PDE
cGMP
2+
Mg
PPi
2+
5'-GMP
Mg
H2O
GC: Guanylate cyclase
(2) Function of PKG
Ser/Thr- P of protein and E
ANP（atrial natriuretic peptides）
↓
GC
NO
↓
cGMP
↓
PKG
↓
Vascular dilatation
peptide hormones
neurotransmitters
AFP
G AC
GC
G PL
GTP
ATP
cAMP
neurotransmitters
hypothalamic pituitrin
Ca2+
PKA
PIP2(PC)
DG
IP3
cGMP
cell memberane
Ca2+-CaM
PKG
PKC
CaM-PK
phosphorylation of enzymes or proteins
trans-acting factor
cis-acting element
nucleus
expression
P
effects
4. Tyrosine-protein kinase
pathway (TPK)
• TPK receptor is related to
proliferation, differentiation,
dissociation, carcinomatous
change.
• TPK：
receptor TPK：Mb.
non receptor TPK：cytosol
(1) Receptor TPK－Ras－MAPK
pathway
• GRB2, SOS, Ras, Raf
• Small G protein: Ras
• MAPK (mitogen-activated protein
kinase)：
MAPK、MAPKK、MAPKKK
EGF, PDGF
receptor
TPK
ÊÜÌå ÐÍTPK
GRB2－P
SOS－P
Ras－GTP
Raf－P
nucleus
trans-acting factor －P
expression
MAPKK－P
MAPK－P
ϸ
°û
Ĥ
(2) JAKs－STAT pathway
ligand
non TPK receptor
JAKs
STAT
gene expression
• JAKs
• STAT: Signal transductors and
activator of transcription
interferons
Plasma membrane
ÖÊĤ
JAK
P£-
£- P
P
84
113
P
91
STAT complex
113
91 84 P
48
inactive STAT
Nuclear
membrane
ºËĤ
P
113
P
84
91
P
48
gene
»ùÒò
Interferons
response
¸ÉÈÅËØ
Ó¦´ð Ôª¼þ element
transcription
Interferon
Dimerization of Receptor
Autophosphorylation of JAK
Phosphorylation of STAT
Nuclear translocation of STAT
Interferon response element
Expression of gene
5. Nuclear factor-κB pathway
6. TGF-βpathway
Intracellular receptor
(DNA transcription regulated
receptor)
• Steroid H, VD3, Thyroxine
• Cytosolic R: glycocorticosteroid H
• Nuclear R: thyroxine, estrogen,
androgen, progesterone
Serum binding protein
With bound bormone