Download Receptor 接受器or受器

Receptor 接受器or受器
Intracellular
receptor
Intercellular signaling in animals by receptors
1
Two classes of receptors have two basic
structural plans in cell membrane:
1. Trimeric G protein-linked receptors (GPCR)
(e.g. glucagon-, serotonin- adrenalin-receptors)
Ionotropic
Metabotropic
2. Ion-channel receptors (ligand-gated ion-channels,
e.g. the acetylcholine receptor) iontropic receptor
G-protein coupled receptor
Tyrosine kinase linked receptor
Instrinsic enzymatic receptor
3. Receptors lacking intrinsic catalytic activity but directly
associated with cytosolic protein tyrosine kinases
4. Receptors with intrinsic enzymatic activity
(e.g. guanylate cyclase activity, protein phosphatase,
serine/threonine kinase or tyrosine kinase activity)
5. Cell adhesion molecules
6. Intracellular receptors
Four classes of cell-surface receptors
1. Ligand-gated ion channels
(ionotropic) exist in cell
membrane. e.g. nAChR
2. G-protein coupled receptors
(metabotropic) exist in cell
membrane e.g. mAChR
3. Tyrosine Kinase linked
Receptor exist in cell
membrane e.g. cytokine
receptors
4. Instrinsic emzymatic
receptor. Receptor Tyrosine
kinase (RTK) can
autophosphorylation.
All receptors are proteins made
up of varying numbers of
subunits or transmembrane
domains.
Cell-surface receptors belong to four major classes
GPCRs are involved in a range of signaling pathways, including
light detection, odorant detection, and detection of certain
hormones and neurotransmitters
Many different mammalian cell-surface receptors including GPCRs
are coupled to a trimeric signal-transducing G protein
– made of an alpha, beta and gamma subunit complex
Ligand binding activates the receptor, which activates the G protein,
which activates an effector enzyme to generate an
intracellular second messenger
– e.g. adenylyl cyclase – converts ATP to cAMP
depending on regulation at the effector enzyme – this pathway can
be either activated or inhibited
– by the type of G protein activated by the hormone-receptor
complex
– Gs proteins result in stimulation of the effector enzyme
– Gi proteins result in inhibition of the effector enzyme
adenylyl cyclase (AC)
2
NO-self enzymatic activity
Four classes of cell-surface receptors
-ligand binding changes the confirmation of the receptor so that specific ions flow
through it the resultant ion movement alters the electric potential across the plasma
membrane
-found in high numbers on neuronal plasma membranes
e.g. ligand-gated channels for sodium and potassium
-also found on the plasma membrane of muscle cells
-binding of acetylcholine results in ion movement and
eventual contraction of muscle
-lack intrinsic catalytic activity
-binding of the ligand results in the formation of a receptor dimer (2 receptors)
-this dimer than activates a class of protein called tyrosine kinases
-this activation results in the phosphorylation of downstream targets by
these tyrosine kinases (stick phosphate groups onto tyrosines within
the target protein)
-receptors for cytokines such as, interferons
Itself enzymatic activity
Signal
transduction
Cascade
-also called receptor tyrosine kinases OR ligand-triggered protein kinases
-similar to tyrosine-linked receptors - ligand binding results in formation of a dimer
-BUT: they differ from tyrosine-linked receptors – intrinsic catalytic activity
-means that ligand binding activates it and the activated receptor acts as a
kinase
-recognize soluble or membrane bound peptide/protein hormones that act as
growth factors
e.g. NGF, PDGF, insulin
-binding of the ligand stimulates the receptor’s tyrosine kinase activity,
-results in phosphorylation of multiple amino acid residues within its target
such as serine and threonine residues
-this phosphorylation activates downstream targets
-its targets are generally other protein kinases –which phosphorylate their
own downstream targets (other kinases??)
3
Itself enzymatic activity
NO-self enzymatic activity
Itself enzymatic activity
Itself enzymatic activity
Itself enzymatic activity
NO-self enzymatic activity
Cell adhesion molecular
4
Four classes of cell-surface receptors
Nucleus or cytoplasmic receptor
G-protein coupled receptors (GPCRs)
Ligand binding activates a G-protein which in turn
activates or inhibits an enzyme that generates a specific
second messenger
estrogen receptor
類酯醇X受體（Pregnane X receptor PXR
How G proteins were discovered
Gilman & Ross studying connection between adrenalin
receptors and the enzyme adenylate cyclase which
makes cyclic AMP
ATP
adenylate cyclase
cAMP
Expt 1
Expt 2
adrenalin
adrenalin
cyc-
+
wild-type extract
(untreated)
cyc-
可能野生種的
AC所導致
cAMP增加
no cAMP
used a mutant cell line cyc- that bound adrenalin but
appeared to lack adenylate cyclase lucky experiment
that led to the Nobel prize!!
cAMP
Expt 2
adrenalin
cyc- +
wild-type extract
(AC inactivated)
把野生種萃取物中AC抑
制掉,但還是有反應?顯示
是cyc- 還是有自己的AC
活性所致, 所以是以前
cyc-對的認知有錯誤,是
少某種物質而非AC
cAMP !?
Explanation: cyc- cells didn’t lack adenylate
cyclase, but lacked another factor (G
proteins) that activates adenylate cyclase
Cyc- mutant ? Not without adenylate cyclase →without some thing
5
The importance of G-proteins
The Nobel Prize in Physiology and Medicine 1994
"for their discovery of G-proteins and the role of these proteins
in signal transduction in cells"
G-protein linked receptors
most common type of receptor
when receptor is activated by stimulus operates via an intermediary
– G protein (guanine nucleotide binding protein)
G protein in turn regulates enzyme or ion channel
all G-protein coupled receptors have 7 transmembrane spanning regions
A major target for drug e.g. beta blockers, antihistamines
Most act via hetero-trimeric G-proteins with cAMP, cGMP and PLC often
being used as downstream effectors
Receptors downregulated following ligand activation to assist with shutoff
of the switch
Alfred G. Gilman
USA
Martin Rodbell
USA
1941-
1925-1998
Largest class of cell surface Receptors
All have a structure with seven transmembrane alpha helical Loops
Orientation is always conserved with N terminus outside, C terminus inside the
cell and cytosolic segments interacting with G proteins
Sequences at C3, C4 and sometimes C2 determine which G protein is activated
Genome sequencing has revealed more new members
Half of all known drugs bind to G protein-linked receptors
Nearly 2500 GPCRs have been identified
Bovine rhodopsin was cloned in 1983 (Nathans and Hogness); adrenergic receptor in 1986 (Dixon et al.)
General structure:
N-terminal segment: glycosylation, ligand binding (outer)
C-terminal segment: phosphorylation and palmitoylation (inner)
Seven transmembrane domain (TMs): form six loops (three
exoloops and three-four cytoloops) and a TM core that could provide
ligand specificity and regulatory mechanism
Membrane bound effector proteins
Effector proteins are then capable of amplifying the signals that will
then be further transduced to secondary targets. Examples:
– Adenylyl cyclase (cAMP synthesis)
– Phospholipase Cβ (inositol lipid release from membrane)
– Phospholipase A2 (arachidonic acid release from membrane, precursor
of prostaglandin and leukotriene synthesis)
– Guanylyl cyclase (cGMP synthesis)
– Cyclic nucleotide phosphodiesterase (breakdown of cGMP and
cAMP)
– Potassium and calcium ion channels Gsα stimulates adenylyl cyclase
GTP bound Gsα interacts with adenylyl cyclase
The structural changes that are induced are not known, but the result is
active enzyme
Many signals, through different receptors, can activate Gsα, resulting in a
higher concentration of GTPGsα and the production of higher levels
of cAMP
Forskolin, applied to cells, will activate pathways mediated by cAMP
adenylyl cyclase activator
6
GPCR encoded for >1000 genes,
represent app.1% of human gemone
GPCRs
-Among membrane-bound receptors,
the G protein-coupled receptors
(GPCRs) are the most diverse.
-In vertebrates, this family contains
1000 – 2000 members (>1% of the genome).
-GPCRs have been very successful during
evolution, being capable of transducing
messages as different as photons, organic
odorants, necleotides, peptides, lipids
and proteins.
-GPCRs have a common ”central core”,
composed of 7 transmembrane helical
domains.
-The fine-tuning of coupling of the receptor
to G proteins is regulated by splicing,
RNA editing and phosphorylation.
Illustration of the central core of
rhodopsin. The core is viewed from
the cytoplasm.
Characteristics of G proteins
1. G protein is an  trimeric protein which binds guanine nucleotides.
2. They function to couple integral membrane receptors to target
membrane-bound enzymes.
3. They can be considered molecular switches wherein…
GDP (inactive)  GTP (active) + 
4. The dissociated  subunit expresses GTPase activity.
(A) GPCRs have a central common core made of seven
transmembrane helices (TM-I to -VII) connected by three
intracellular (i1, i2, i3) and three extracellular (e1, e2, e3) loops.
The diversity of messages which activate those receptors is an
illustration of their evolutionary success. (B) Illustration of the
central core of rhodopsin. The core is viewed from the
cytoplasm.
cytoplasm The length and orientation of the TMs are
deduced from the two-dimensional crystal of bovine and frog
rhodopsin (Unger et al., 1997). The N- and C-terminal of i2
(including the DRY sequence) and i3 are included in TM-III and
-VI. The core is represented under its 'active conformation'.
The TM-VI and -VII lean out of the structure, the TM-VI turn by
30% on its axis (clockwise as viewed from the cytoplasm)
(Bourne, 1997). This opens a cleft in the central core in which
G proteins can find their way. i2 and i3 loops are the two main
loops engaged in G protein recognition and activation.
EMBO J. 18: 1723-1729 (1999)
GPCRs
5. GTPS blocks GTPase
activity of GTP.
7
Receptor activation…
GPCRs activate different sub-classes of heterotrimeric
G-proteins and effector systems (cont’d)
Heterotrimeric G-proteins
Highly conserved mechanism linking to most GPCR’s
Following activation of GPCR the GDP on G( heterotrimeric Gprotein is switched for GTP causing dissociation to G and G after
stimulation. Both can have signalling effects
G Over 20 forms have now been identified in mammals. Divided into
Gs which generally activate effector, Gi which generally inhibitory,
Gq which generally act via phospholipases. The GTPase activity acts
as a time dependent switch by converting GTP to GDP
G About 6 forms of  and 12 forms of  have now been identified. The
G stay as dimer and can regulate molecules including K+ channels
and PI 3-kinases
Nature Reviews Molecular Cell Biology 3; 639-650
Receptors cycle between resting and active states
G proteins are activated in response to binding by an activated receptor
General Themes in Heterotrimeric
G protein Pathways
May be > 20
GTP displaces GDP and subunits dissociate. Gα is activated
Rockman H.A. et al (2002)
Nature 415:206
8
The tools for G-protein receptor signal transduction research
So, inhibitory signal ↓
So, stimulatory signal ↑
Basic structure/function of G-protein subunits:
heterotrimers consist of one copy of alpha (39-45kDa), beta
(35-36kDa) and gamma (5-7kDa) subunits
G-protein ADP ribosylation
+
Nicotinamide
CTX: for Gs
PTX: for Gi
Gs
Bacterial toxin
(PTX, CTX)
ADP-ribose
G
GTPase
9
Family 1 contains most GPCRs
including receptors for odorants (氣
味), small ligands, peptide hormone
and glycoprotein. Disulphide bridge
connects e1 and e2 and palmitoylated
cysteine in C-terminal.
Family 2 GPCRs have relative long Nterminal that contains several
cysteines (network of disulphide
bridge). Examples include glucagon,
GnRH and PTH receptors.
Family 3 GPCRs have very large span
N-terminal sequences and C-terminal
tail. The ligand binding domain is
located in the N-treminus. The i3 loop
is short and highly conserved.
Representative samples are mGluR,
Ca2+-sensing and GABA-B receptor.
85 kDa
Functional regions within GPCRs: G-protein interacting
domains & ligand binding domain
The three subfamilies of
一種味覺
GPCRs are depicted with
examples of their
endogenous agonists.
The binding modes of the
orthosteric (直立) ligands
for each receptor type
are depicted by a green
rectangle. The GPCR
signals either by coupling
to heterotrimeric Gproteins consisting
of and
subunits
(which trigger a wide
range of metabolic
cascades and ion
channel activities) or by
direct association with
effector molecules. AC,
adenylyl cyclase; ATP,
adenosine triphosphate;
cAMP, cyclic adenosine
monophosphate; PLC,
phospholipase C; IP3,
inositol-3,4,5-trisphosphate; DAG,
diacylglycerol.
Schematic presentation of the general structure of GPCRs
and receptorreceptor-ligand interactions
1. Proteinase-activated receptor ( PAR ) Family
PAR1~4
2. PAR2’s activation induces acute inflammation
Platelet aggregation
J Biol Chem 273:17299-17302,1998
10
Ligand Binding and GPCR Activation
The G subunit of G protein cycles between active and
inactive forms
• Modes of ligand binding:
Exclusive in TM core: photon, biogenic amines, nucleotides
and lipid substrates (B)
core, exoloops and N-terminal segment: peptides of ≤40
amino acids (C)
N-terminal segment cleavage: protease( thrombin) (D)
N-terminal segment and exoloops: glycoprotein hormone
( approximately 350 amino acids) (E)
N-terminal segment (~600 amino acids): calcium channel,
GABA and metabotropic receptor (F)
FRET
Fluorescence resonance energy transfer
Applications for monitoring molecular interactions in
Living cells by FRET
Monitor protein-protein interaction
Calcium sensor
Monitor intramolecular conformational change
Kinase activation sensor
Receptor mediated activation of coupled G-proteins occurs within
a few seconds of ligand in living cells
M13, a peptide binds to calmodulin in
calcium-dependent manner
11
Diversity (of physiological responses to GPCR stimulation)
Receptor activation…
GPCRs activate different
sub-classes of heterotrimeric
G-proteins and effector systems
GRK: G-protein coupled receptor kinase
GPCR
↓
Second messenger
↓
effector
↓
response
TARGET TISSUE
HORMONE
MAJOR RESPONSE
Thyroid gland
thyroid-stimulating hormone
(TSH)
thyroid hormone synthesis and
secretion
Adrenal cortex
adrenocorticotrophic hormone
(ACTH)
cortisol secretion
Ovary
luteinizing hormone (LH)
progesterone secretion
Muscle
adrenaline
glycogen breakdown
Bone
parathormone
bone resorption
Heart
adrenaline
increase in heart rate and force
of contraction
Liver
glucagon
glycogen breakdown
Kidney
vasopressin
water resorption
Fat
adrenaline, ACTH, glucagon,
TSH
triglyceride breakdown
TARGET TISSUE
SIGNALING MOLECULE
MAJOR RESPONSE
Liver
vasopressin
glycogen breakdown
Pancreas
acetylcholine
amylase secretion
Smooth muscle
acetylcholine
contraction
Blood platelets
thrombin
aggregation
GPCR vs. RTK
Complexity of GPCR signalling
Cascades
GPCRs cross talk with Receptor
Tyrosine Kinases (RTK)
Given such a diversity in
responses, how does GPCR
signaling specificity occur???
AC: adenylyl cyclase
PDE: phosphodiesterase
PLC: phospholipase C
Multiple physiological responses
12
The mechanisms regulate (terminate) signaling form GPCR
1.GTP → GDP (exchange)
2. Degradation of second message, cAMP phosphodiesterase
(cAMP →5’AMP) or cGMP phosphodiesterase…
3.Receptor phophorylation by down stream signal (cAMP → PKA
→ phosphorylation of receptor); feedback regulation
(Desentization; heterologus or homologous)
4. Protein Phosphatase catalyzes removal by hydrolysis of
phosphates that were attached to proteins via Protein Kinase A
Turn off of the signal from GPCR
Agonist or antagonist → receptor → activation of receptor specific
enzyme (receptor kinase) → phosphorylation; directly from receptor
action is called homologus desentization
Anchoring proteins localize effects of cAMP to specific subcellular
regions
(new model for turn off signal from GPCR)
Phosphodiesterase enzymes
catalyze:
cAMP + H2O  AMP
N
N
The phosphodiesterase that
cleaves cAMP is activated by
phosphorylation catalyzed by
Protein Kinase A.
N
N
H2
5' C
O
Thus cAMP stimulates its
own degradation, leading to
rapid turnoff of a cAMP signal.
NH2
cAMP
O
4'
O
H
H 3'
P
O
O-
H
1'
2' H
OH
In heart muscle: adrenergic receptor → cAMP ↑→ activate PKA → C,
catalytic region → phosphorylate → PDE → activation → degradation
of cAMP
PDE:phosphodiesterase, cAMP →5’AMP
AKPA: ANCHORING PROTEIN
13
AKAPs (A-Kinase Anchoring Proteins) are scaffold
proteins with multiple domains that bind to
 regulatory subunits of Protein Kinase A
 phosphorylated derivatives of phosphatidylinositol
 various other signal proteins, such as:
• G-protein-coupled receptors (GPCRs)
• Other kinases such as Protein Kinase C
• Protein phosphatases
• Phosphodiesterases
AKAPs localize hormone-initiated signal cascades within a
cell, and coordinate activation of protein kinases as well as
rapid turn-off of such signals.
Receptor desensitization occurs. This process varies with the
hormone.
Some receptors are phosphorylated via specific receptor
kinases.
The phosphorylated receptor may then bind to a protein
arrestin, that promotes removal of the receptor from the
membrane by clathrin-mediated endocytosis.
First discovery: -adrenergic receptor
Heterologous desensitization:
– Four residues in the cytosolic domain of the adrenergic receptor can be phosphorylated by PKA
– Activity of all Gs protein – coupled receptors, not just
the -adrenergic receptor, is reduced
Homologous desensitization:
– Other residues in the cytosolic domain of the adrenergic receptor are phosphorylated by the receptorspecific -adrenergic receptor kinase (BARK)
– BARK only phosphorylates the -adrenergic receptor
which facilitates -arrestin binding to the phosphorylated
receptor
– Related with GRK (G-protein receptor kinase
Hormonally induced negative
regulation of receptors is
referred to as homologousdesensitization
This homeostatic mechanism
protects from toxic effects of
hormone excess.
Heterologous desensitization
occurs when exposure of the
cell to one agonist reduces the
responsiveness of the cell any
other agonist that acts through a
different receptor.
This most commonly occurs
through receptors that act
through the adenylyl cyclase
system.
Heterologous desensitization
results in a broad pattern of
refractoriness with slower onset
than homologous
desensitization
14
G protein-coupled receptor kinases (GRKs)
Receptor phosphorylation by second messenger kinases
A family of serine-threonine
kinases that recognizes
and phosphorylates
receptors in their
agonist-stimulated
form
Consensus sequence:
serine/threonine
residues surrounded by
acidic residues
DLEESSSSD
Members of the GRK family and their regulation
GRK
Tissue expression
Regulation
GRK1 (rhodopsinkinase)
Retinal rods and cones
Farnesylation of C terminus
GRK2 (ARK1)
Ubiquitous, brain
PIP2 and G binding
GRK3 (ARK2)
Ubiquitous, in the brain
PIP2 and G binding
is lower than GRK2
Heterologous desensitization
GRK4
Testes; low in brain
GRK5
Ubiquitous, brain
Modulated by CAM and calcium sen
sor proteins
GRK6
Ubiquitous, brain
palmitoylated cysteine residues
GRK7
Retinal cones
Geranygeranylated
Receptor phosphorylation by GRKs
到處存在
GRK: G-protein
receptor kinase
homologous-desensitization
Regulation of GRK function
PIP2-binding through polybasic C ter
minus domains and palmitoylated
cysteine residues
GRK: G-protein coupled receptor kinase
Desensitization or Endocytosis of GPCR’s
Effected by Phosphorylation
1. The ligand activated receptor can be phosphorylated on select Ser/Thr residues by
GRK (e.g. BARK -  adrenergic receptor kinase). These phosphorylated residues
provide a docking site for arrestin resulting in inactivation/desensitization.
2. In some instances, arrestin binding targets the receptor for clathrin-dependent
endocytosis.
3. In addition, if the occupied GPCR leads to  cAMP, the receptor can also be
phosphorylated by PKA leading to its inactivation/densensitization.
Penela P, Ribas C, Mayor F. Jr. Mechanisms of regulation of the expression and
function of G protein-coupled receptor kinases. Cell Signal. 2003 Nov;15(11):973-981.
15
Clathrin-dependent Receptor-mediated endocytosis
-Arrestins : intracellular protein
- Interaction with phosphorylated GPCRs uncouples the
receptors from
heterotrimeric G proteins, producing a nonsignaling,
desensitized receptor. (desensitization )
- Target the GPCRs to clathrin-coated pits for
endcytosis to function as docking proteins that link
receptors to components of the endocytic machinery
such as AP-2 and clathrin (internalization )
- Regulate the dephosphorylation of Receptors
(resensitization).
GRK-phosphorylation/
arrestin binding : uncouling,
desensitization
Clathrin coated pit :
pinch off –dynamin
- sequestration
Agonist binding
?
The ability of -Arrestins to remain associated with some receptors
but not others suggests that -Arrestins may regulate the cellular
trafficking and dephosphorylation of receptor and ultimately their
kinetics of resensitization.
Ref. Seminars in Cell & Developmental biology 9,1998
Receptor Down-Regulation
NO-self enzymatic activity
Slower onset (hours to days), more prolonged effect
Decreased synthesis of receptor proteins
Increase in receptor internalization and degradation
Internalization involves endocytosis of receptor: the endocytic
vesicle may ultimately return the receptor to the cell surface, or
alternatively may deliver the receptor to a lysosome for
destruction.
Endocytic vesicles are associated with phosphatases which can
clear phosphate from a receptor and ready it for reuse before
returning it to the plasma membrane.
-lack intrinsic catalytic activity
-binding of the ligand results in the formation of a receptor dimer (2 receptors)
-this dimer than activates a class of protein called tyrosine kinases
-this activation results in the phosphorylation of downstream targets by
these tyrosine kinases (stick phosphate groups onto tyrosines within
the target protein)
-receptors for cytokines such as, interferons
16
G protein-coupled receptors transmit signals to MAP kinase
Two types of intracellular signaling complexes
Activated G of Gprotein May also
InduceMAPK Cascade
Scaffold protein
All MAP kinase are
serine/threonine kinase
Adapter protein: directly contact with receptor
Cytokine receptor
Two types of intracellular signaling complexes
All molecule attached at scaffold protein
Cytokine receptors signal to the nucleus in a direct
pathway
Tyrosine kinase receptors
One protein → phosphorylation → induced another protein bind → induced another protein
17
Signaling pathway using modular binding domains
Signal protein complexes (more efficiency and specific)
Signal cascades are often mediated by large "solid state" assemblies that
may include receptors, effectors, and regulatory proteins, linked
together in part by interactions with specialized scaffold proteins.
Scaffold proteins often interact also with membrane constituents,
elements of the cytoskeleton, and adaptors mediating recruitment into
clathrin-coated vesicles.
They improve efficiency of signal transfer, facilitate interactions among
different signal pathways, and control localization of signal proteins
within a cell.
One strategy the cell uses to achieve specificity involves scaffolding proteins
They organize groups of interacting signaling proteins into signaling
complexes
Because the scaffold guides the interactions between the successive
components in such a complex, the signal is relayed with speed
Adapter protein: directly
contact with receptor; It
also signal players
In addition, cross-talk between signaling pathways is avoided
Scaffolding proteins are large relay proteins to which other
relay proteins are attached
Scaffolding proteins can increase the signal transduction
efficiency
Signal
molecule
Plasma
membrane
Some receptors and signal transduction protein are localized
Clustering of membrane proteins mediated by adapter domains
Synaptic junction: chemical signal → presynaptic cell → clustering receptor →
raid and efficient signal transmission
PDZ: 90 a.a.; target protein Ser-Thr-X-Φ; X: any, Φ: hydorphobic
PDZ interact with subunit receptor formed complex
Receptor
Three
different
protein
kinases
Scaffolding
protein
Protein motifes
(1) Mediate protein-protein interactions
(2) Determine the location of signaling proteins
18
Different kinds of intracellular signaling proteins along a
signaling pathway from the cell surface to the nucleus
Signal Transduction Domains
Src tyrosine kinase contains 4
functional regions (known function
and by homologies with domains in
other proteins).
The Src Homology (SH) domains
have subsequently been defined as
-SH1 being the Tyrosine Kinase
-SH2 being a domain that binds
phosphorylated tyrosines (or PTB)
-SH3 being a domain that binds
proline rich regions
-SH4 being a domain that regulated
addition of lipids
Thus the domains contain not just
enzyme activities but control the
formation of protein complexes and
anchoring to membranes
signal
receptor
Lipid eg PIP3
PH
PP
Tyr K
YP
SH2
P
SH3
Relay(接替) proteins: pass the message
to the next signaling component
Adaptor proteins: link one signaling
protein to another without themselves
participating in the signaling event
Bifurcation (分枝)proteins: spread the
signal from one signaling pathway to
another
Amplifier proteins: usually either
enzymes or ion channels that enhance
the signal they receive
PPP
effects
Ligand binding to the receptor activates an intrinsic
enzymatic activity
Receptors with intrinsic
enzymatic activity
(e.g. guanylate cyclase
activity, protein phosphatase,
serine/threonine kinase or
tyrosine kinase activity)
Transducer proteins: convert the signal
to a different form e.g. adenyl cyclase
Tyrosine kinase receptors
these receptors traverse the membrane only once
receptor has intrinsic enzyme activity
– i.e. the receptor itself is an enzyme
respond exclusively to protein stimuli
– cytokines
– mitogenic growth factors:
• platelet derived growth factor
• epidermal growth factor
These usually receive signals that regulate: cell proliferation (growth
factors) or cell differentiation (inducers)
 Tyrosine kinase activation is key event in intracellular signal transduction.
 Most Tyrosine Kinase Receptors exist as inactive monomers in membrane.
 Receptor autophosphorylation occurs via a ligand-induced receptor
dimerization
 Phosphotyrosine residues in cytoplasmic domain of receptor act as docking
sites that bind cytoplasmic signaling molecules.
19
There are six major families of receptor tyrosine kinases.
All have a TK domain on the cytosolic, COOH-terminal end,
A single-pass transmembrane domain, and
One or more cysteine-rich or Immunoglobuliu-like ligand-binding domains.
Tyrosine kinase:
(phosphorylation & dephosphorylation)
kinase enzymes add a phosphate (Pi) group
tyrosine kinase speicfically to tyrosine residue
phosphatases remove Pi
phosphorylation state alters shape (conformation) of protein and
changes its function
– enzyme activity; solute transport; gene expression
covalent modification by phosphorylation is extremely important
in regulation biological responses
•
An individual tyrosine-kinase receptor
consists of several parts:
– an extracellular signal-binding sites,
– a single alpha helix spanning the
membrane, and
– an intracellular
tail with several
tyrosines.
•
•
•
When ligands bind to two receptor
polypeptides, the polypeptides
aggregate, forming a dimer.
This activates the tyrosine-kinase
section of both.
These add phosphates to the tyrosine
tails of the other polypeptide.
•
•
The fully-activated receptor
proteins activate a variety of
specific relay proteins that bind
to specific phosphorylated
tyrosine molecules.
– One tyrosine-kinase
receptor dimer may
activate ten or more
different intracellular
proteins simultaneously.
These activated relay
proteins trigger many
different transduction
pathways and
responses.
20
Tyrosine Kinase Receptors
Tyrosine Kinase Receptors
Note steps involved:
1. Ligand Reception
2. Receptor Dimerization
3. Catalysis (Phosphorylization)
4. Subsequent Protein Activation
5. Further Transduction
6. Response
The TGFβ superfamily
consists of many members.
Bone morphogenetic
proteins (BMP) is the
largest family.
TGFβ is formed by cleavage
of a secreted inactive
precursor
During development, TGFβ
signaling is involved in
pattern formation, cell
proliferation,
differentiation, ECM
production, and cell death.
In adults, TGFβ is involved
in tissue repair and immune
regulation.
Maturation of TGFβ is
dependent on release from
LTBP (latent TGF- –
binding protein) by
proteolysis.
TGF- receptor and the direct activation of Smads
潛在
RII receptor has a constitutive
ser/thr kinase activity
TGFβ binding induces complex
formation between RII and RI;
phosphorylation of RI by RII
activates RI kinase activity
RI did not bind to TGF
RI kinases Smad transcription
factors. Phosphorylation results in
a conformational change in Smad
phosphorylation
Complexes with other transcriptional
factor = Smad4
Complex moves in nucleus
Activates gene
Gene = plasminogen activator
inhibitor  no cell growth
TGFR: receptor serine/
threonine kinase
Smad: transcription factor
TFE: transcription factor
21
Membrane Form of Guanylyl Cyclase
Atrial natriuretic factor
Small hydrophobic signaling molecules, such
as steroids, can cross the cell membrane
(e.g. estrogen, vitamin D, thyroid hormone,
retinoic acid) and bind to intracellular
receptors
1. Receptor guanylyl cyclases
generate cGMPdirectly as an
intracellular mediator
2. Atrial natriuretic peptides
(ANPs) are family of related
peptide hormones
3. Single pass
transmembrane protein that
has extracellular binding site
for ANPs and an intracellular
guanylyl cyclase catalytic unit.
4. Binding of ANP activates
cylase to produce cGMP
which in turn activates cGMPdependent protein kinase (GKinase)
kidney
Intracellular receptors (nucleus receptor)
intestinal epithelial cells
The hormone-receptor complex has an
exposed DNA binding site and can
activate transcription directly (or, more
typically as a homo- or hetero-dimer)
This usually initiates a cascade of
transcription events
GTP
cGMP
Nuclear Receptors
Some signaling molecules that
bind to intracellular receptors
Lipid soluble ligands that penetrate cell membrane (corticosteroids,
mineralocorticoids, sex steroids, Vitamin D, thyroid hormone)
Receptors contain DNA-binding domains and act as ligand-regulated
transcriptional activators or suppressors(=> characteristic lag period of
30 minutes to several hours):
cortisol
response
Ligand binding of the receptors triggers the
formation of a dimeric complex that can
interact with specific DNA sequences
(=“Response Elements”) to induce
transcription. The resulting protein
products possess half-lifes that are
significantly longer than those of other
signaling intermediates => Effects of
nuclear receptor agonists can persist for
hours or days after plasma concentration
is zero.
雌二酮
estradiol
睪固酮
testosterone
Vitamin D3
一種腎上腺
皮質內泌素
thyroxine
甲狀腺素
Retinoic acid
維他命Ａ酸
22
Nuclear Receptors
•
The nuclear receptor superfamily
Examples:
– Glucocorticoids: Inhibit transcription of COX-2; induce transcription
of Lipocortin
– Mineralcorticoids: Regulate expression of proteins involved in renal
function
– Retinoids (Vit A derivatives): Control embryonic development of
limbs and organs; affect epidermal differentiation => dermatological
use (Acne)痤瘡 粉刺
– PPARs (Peroxisome Proliferation-Activated Receptors): control
metabolic processes:
• PPAR: Target of Fibrates (cholesterol lowering drugs: stimulate
-oxidation of fatty acids)
• PPAR: Target of Glitazones (anti-diabetic drugs: induce
expression of proteins involved in insulin signaling => improved
glucose uptake)
Hinge 鉸縺
23
Nuclear Receptor Family
is Large but not ubiquitous:
mammals have ~50-60 genes
flies 21
worms 270 (!!!)
plants 0
yeast 0
Only a handful of physiological ligands have
been identified,
(despite many genes, worms lack any known
lipid based endocrine system)
Early primary response (A) and delayed secondary
response (B) that result from the activation
of an intracellular receptor protein
Nuclear receptor family (steroid)
Steroid hormone receptors are part of the superfamily of nuclear
receptors that contains over 30 members.
All members have conserved regions of high homology
Hormone binding domain 90% homologous
10% difference accounts for specificity
DNA binding domain which contains zinc fingers
Receptors are found complexed with heat shock proteins (HSP)
Unoccupied receptor held in inactive conformation by HSP
Ligand binding releases HSP and exposes DNA binding domain
Hormone receptor complex then binds to response elements on
gene and allows transcription to occur
Ligand gate Ion-channel receptors
Ligand binding changes the conformation of the
receptor so that specific ions flow through it
24
Nongated ion channels and the resting membrane potential
Types of Membrane Ionic Channels
Non-gated channels: leakage channels open at rest
Gated: need ligand to activation; Non-gated: do not need ligand
Ion Channel (non-gate)
Generation of electrochemical gradient across plasma membrane
Gated Channels:
– Voltage-gated channels
– Mechanically-gated channels
– Chemically-gated channels (from outside or inside of
the membrane)
• Neurotransmitter-activated
• Calcium-gated
• ATP-gated
• Cyclic nucleotide-gated
• About 100 different kinds of channels
Ligand gate Ion channel characterizations
multi-subunit, transmembrane protein complexes
complex is both the receptor and ion channel
stimuli: chemical, stretch, voltage or light
stimulus induces conformational change to open or close ion channel
i.e. Ca+ gradient
regulation of signal transduction , muscle
contraction and triggers secretion of digestive
enzyme in to exocrine pancreastic cells
i.e. Na+ gradient
uptake of a.a , symport, antiport; formed membrane potential
i.e. K+ gradient
formed membrane potential
Q: how does the electrochemical gradient formed?
Selective movement of Ions Create a
transmembrane electric potential difference
Ligand-gate ion channels
chemical stimuli bind to receptor and open or close ion channel
stimuli can be extracellular or intracellular
EXTRACELLULAR STIMULI: (neurotransmitters)
– e.g. acetylcholine, dopamine, GABA, glutamate
INTRACELLULAR STIMULI: (second messengers)
– e.g. IP3, cAMP, cGMP, Ca2+

Light gated channels
respond to light; in the eyes

Mechanically gated
channels respond to vibration
or pressure - in the ear, touch
25
NMDA receptor-Ligand gated channel
Ion-channel-linked receptors
Convert chemical signals ==> electrical signals
Extracellular ligand-gated
nicotinic ACh (muscle): 2 (embryonic), 2 (adult)
nicotinic ACh (neuronal): (2-10), (2-4)
glutamate: NMDA, kainate, AMPA
P2X (ATP)
Many other types of transmembrane ion channels
==> Ion channels are common drug targets!
• Voltage-gated channels:
• Gating: controlled by membrane
polarization/depolarization
• Selectivity: Na+, K+ or Ca+ ions
5-HT3
GABAA: (1-6), (1-4),  (1-4), , , (1-3)
Glycine
• Intracellular ligand-gated channels:
• Ca+ controlled K+ channel
• ATP-sensitive K+ channel
• IP3-operated Ca+ channel (in the ER
membrane)
26
Voltage gate ion channels
ion channel undergoes conformational change folllowing electrical stimulus
this “depolarization” opens the channel
– leads to flow of Na+ into cell
– constitutes an “action potential”
channel re-closes
Pseudo-subunit vs. true subunit structure
Passive-Mediated Transport
• Gated vs non-gated
• Gated
Na+-channel
Non-Gated
- voltage
Na+-channel
Na+-channel - chemical
K+-channel - voltage
K+-channel
K+-channel - chemical
Cl--channel - chemical
Cl--channel
27
Regulation of Ion Channels
A wide range of plasma membrane ligand and voltage sensitive ion
channels exist controlling cytoplasmic levels of Na+, K+, and Ca2+.
For example, acetyl choline receptor allows influx of Na+ and K+ triggering
action potential in nerve/muscle
Cytoplasmic Ca2+ levels regulated via outer membrane- and ER
receptors (ryanodine and IP3)
Changes in level of Ca2+ are particularly dramatic (1000 fold increase)
A major effector of Ca2+ is calmodulin which activates myosin light chain
kinase (hence promotes contraction) and calmodulin dependent
protein kinase (metabolism, transcription etc)
ATP dependent pumps rapidly transport Ca2+ back to where it came from
meaning the signal can be very rapidly shut off then switched on
again – eg muscle contraction
Intracellular ligand-gated
leukotriene C4-gated
Ca2+
ryanodine receptor Ca2+
IP3-gated Ca2+
IP4-gated Ca2+
Ca2+-gated K+
Ca2+-gated nonselective cation
Ca2+-gated Cl–
cAMP cation
cGMP cation
cAMP chloride
ATP Cl–
volume-regulated Cl–
arachidonic acidactivated K+
Na+-gated K+
G-protein linked receptors
coupled to ion channels
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Acetylcholine (muscarinic)
•
Adenosine & adenine nucleotides •
Adrenaline & noradrenaline
•
Angiotensin
•
Bombesin
•
Bradykinin
•
Calcitonin
•
Cannabinoid
•
Chemokine
•
Cholecystokinin & gastrin
•
Dopamine
•
Endothelin
•
Galinin
•
GABA (GABAB)
•
Glutamate (quisqualate)
•
Histamine
5-Hydroxytryptamine (1,2)
Leukotriene
Melatonin
Neuropeptide Y
Neurotensin
Odorant peptides
Opioid peptides
Platelet-activating factor
Prostanoid
Protease-activated
Tachykinins
Taste receptors
VIP
Vasopressin and oxytocin
28