1. Trimeric G protein-linked receptors (GPCR) (e.g. glucagon-, serotonin- adrenalin-receptors) 2. Ion-channel receptors (ligand-gated ion-channels, e.g. the acetylcholine receptor) iontropic receptor

Receptor 接受器or受器

Intracellular receptor

Intercellular signaling in animals by receptors

1. Trimeric G protein-linked receptors (GPCR) (e.g. glucagon-, serotonin- adrenalin-receptors) 2. Ion-channel receptors (ligand-gated ion-channels, e.g. the acetylcholine receptor) iontropic receptor

3. Receptors lacking

intrinsic catalytic activity

but directly directly

associated with cytosolic protein tyrosine kinases

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

Two classes of receptors have two basic structural plans in cell membrane:

G-protein coupled receptor Tyrosine kinase linked receptor Instrinsic enzymatic receptor

Ionotropic Metabotropic

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.

Four classes of cell-surface receptors 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 stimulationof the effector enzyme – Gi proteins result ininhibitionof the effector enzyme

adenylyl cyclase (AC)

Four classes of cell-surface receptors

-ligand binding changes the confirmationof the receptor so that specific ions flow through it the resultant ion movement alters the electric potentialacross 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

NO-self enzymatic activity

-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??)

Signal transduction Cascade

Itself enzymatic activity

Itself enzymatic activity

Itself enzymatic activity

Itself enzymatic activity NO-self enzymatic activity Itself enzymatic activity

NO-self enzymatic activity

Cell adhesion molecular

Nucleus or cytoplasmic receptor

estrogen receptor 類酯醇X受體（Pregnane X receptor PXR

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

Four classes of cell-surface receptors

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

used a mutant cell line cyc

that bound adrenalin but appeared to lack adenylate cyclase lucky experiment that led to the Nobel prize!!

cyc

adrenalin Expt 1

no cAMP

cyc^-

+ wild-type extract

(untreated)

adrenalin Expt 2

cAMP

adrenalin

+

wild-type extract (AC inactivated)

Expt 2

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

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 可能野生種的

AC所導致 cAMP增加

The importance of G-proteins

The Nobel Prize in Physiology and Medicine 1994

Alfred G. Gilman Martin Rodbell

USA USA

1941- 1925-1998

"for their discovery of G-proteinsand 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

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-terminalsegment: glycosylation, ligand binding (outer) C-terminalsegment: 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

Largest class of cell surface Receptors

All have a structure with seven transmembranealpha helical Loops

Orientation is always conserved with N terminus outside, C terminus insidethe 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

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

GPCRs

Illustration of the central core of rhodopsin. The core is viewed from the cytoplasm.

-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.

(A) GPCRs have a central commoncore 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 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) GPCR encoded for >1000 genes, represent app.1% of human gemone

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…



(inactive)  

(active) + 

4. The dissociated  subunit expresses GTPase activity.

Characteristics of G proteins

5. GTPS blocks GTPase activity of GTP.

GPCRs

Nature Reviews Molecular Cell Biology 3; 639-650 Receptor activation…

GPCRs activate different sub-classes of heterotrimeric G-proteins and effector systems (cont’d)

Heterotrimeric G-proteins

Highly conserved mechanismlinking to most GPCR’s

Following activation of GPCR the GDP on G( heterotrimeric G- protein 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

Receptors cycle between resting and active states

G proteins are activated in response to binding by an activated receptor

GTP displaces GDP and subunits dissociate. Gα is activated

General Themes in Heterotrimeric G protein Pathways

Rockman H.A. et al (2002) Nature 415:206

May be > 20

Basic structure/function of G-protein subunits:

heterotrimers consist of one copy of alpha (39-45kDa), beta (35-36kDa) and gamma (5-7kDa) subunits

The tools for G-protein receptor signal transduction research

So, inhibitory signal ↓ So, stimulatory signal ↑

G-protein ADP ribosylation

+ G_s

Bacterial toxin (PTX, CTX) Nicotinamide

ADP-ribose G

GTPase

CTX: for Gs

PTX: for Gi

85 kDa

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 N- terminal 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.

The three subfamilies of 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 G- proteins 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-tris- phosphate; DAG, diacylglycerol.

Functional regions within GPCRs: G-protein interacting domains & ligand binding domain

一種味覺

Schematic presentation of the general structure of Schematic presentation of the general structure of GPCRsGPCRs

and receptor

and receptor--ligandligandinteractionsinteractions

J Biol Chem 273:17299-17302,1998 Platelet aggregation

1. Proteinase-activated receptor ( PAR ) Family PAR1~4

2. PAR2’s activation induces acute inflammation

Ligand Binding and GPCR Activation

• 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)

The G subunit of G protein cycles between active and inactive forms

Receptor mediated activation of coupled G-proteins occurs within a few seconds of ligand in living cells

FRET

Fluorescence resonance energy transfer

Applications for monitoring molecular interactions in Living cells by FRET

Monitor protein-protein interaction Monitor intramolecular conformational change

Calcium sensor

M13, a peptide binds to calmodulin in

Kinase activation sensor

Receptor activation…

GPCRs activate different sub-classes of heterotrimeric G-proteins and effector systems

GPCR

↓ Second messenger

↓ effector

↓ response

GRK: G-protein coupled receptor kinase

triglyceride breakdown adrenaline, ACTH, glucagon,

TSH Fat

water resorption vasopressin

Kidney

glycogen breakdown glucagon

Liver

increase in heart rate and force of contraction

adrenaline Heart

bone resorption parathormone

Bone

glycogen breakdown adrenaline

Muscle

progesterone secretion luteinizing hormone (LH)

Ovary

cortisol secretion adrenocorticotrophic hormone

(ACTH) Adrenal cortex

thyroid hormone synthesis and secretion

thyroid-stimulating hormone (TSH)

Thyroid gland

MAJOR RESPONSE HORMONE

TARGET TISSUE

aggregation thrombin

Blood platelets

contraction acetylcholine

Smooth muscle

amylase secretion acetylcholine

Pancreas

glycogen breakdown vasopressin

Liver

MAJOR RESPONSE SIGNALING MOLECULE

TARGET TISSUE

Diversity(of physiological responses to GPCR stimulation)

AC: adenylyl cyclase PDE: phosphodiesterase PLC: phospholipase C

Complexity of GPCR signalling Cascades

Multiple physiological responses GPCRs cross talk with Receptor Tyrosine Kinases (RTK)

Given such a diversity in responses, how does GPCR signaling specificity occur???

GPCR vs. RTK

Turn off of the signal from GPCR

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 Agonist or antagonist → receptor → activation of receptor specific enzyme (receptor kinase) → phosphorylation; directly from receptor action is called homologus desentization

Phosphodiesterase enzymes catalyze:

cAMP + H

O  AMP The phosphodiesterase that cleaves cAMP is activated by phosphorylation catalyzed by Protein Kinase A.

Thus cAMP stimulates its own degradation, leading to rapid turnoff of a cAMP signal.

N

N N

N NH₂

O

O OH H H

H H₂

C O H

P

O O-

1' 3'

5' 4'

2'

cAMP

Anchoring proteins

localize effects of cAMP to specific subcellular regions

(new model for turn off signal from GPCR)

PDE:phosphodiesterase, cAMP →5’AMP AKPA: ANCHORING PROTEIN

In heart muscle: adrenergic receptor → cAMP ↑→ activate PKA → C, catalytic region → phosphorylate → PDE → activation → degradation of cAMP

AKAPs

(A-Kinase Anchoring Proteins) are scaffold

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

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

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 receptor- specific -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 homologous- desensitization

This homeostatic mechanism protectsfrom 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

G protein-coupled receptor kinases (GRKs)

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

Receptor phosphorylation by second messenger kinases

Receptor phosphorylation by GRKs

Heterologous desensitization

homologous-desensitization GRK: G-protein

receptor kinase

GRK Tissue expression Regulation

GRK1 (rhodopsinkinase) Retinal rods and cones Farnesylation of C terminus GRK2 (ARK1) Ubiquitous, brain PIP₂and G binding GRK3 (ARK2) Ubiquitous, in the brain PIP₂and G binding

is lower than GRK2

GRK4 Testes; low in brain PIP₂-binding through polybasic C ter minus domains and palmitoylated cysteine residues

GRK5 Ubiquitous, brain Modulated by CAM and calcium sen

sor proteins

GRK6 Ubiquitous, brain palmitoylated cysteine residues

GRK7 Retinal cones Geranygeranylated

Members of the GRK family and their regulation

GRK: G-protein coupled receptor kinase 到處存在

Regulation of GRK function

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.

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.

-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).

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.

Clathrin-dependent Receptor-mediated endocytosis

Ref. Seminars in Cell & Developmental biology 9,1998 Agonist binding

GRK-phosphorylation/

arrestin binding : uncouling,

desensitization Clathrin coated pit :

pinch off –dynamin - sequestration

?

Receptor Down-Regulation

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

NO-self enzymatic activity

Two types of intracellular signaling complexes

Adapter protein: directly contact with receptor

Cytokine receptor

G protein-coupled receptors transmit signals to MAP kinase

Activated G_ of G- protein May also InduceMAPK Cascade Scaffold protein All MAP kinase are serine/threonine kinase

All molecule attached at scaffold protein

Two types of intracellular signaling complexes Tyrosine kinase receptors

One protein → phosphorylation → induced another protein bind → induced another protein

Cytokine receptors signal to the nucleus in a direct

pathway

Signaling pathway using modular binding domains

Adapter protein: directly contact with receptor; It also signal players

Signal protein complexes (more efficiency and specific)

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 In addition, cross-talk between signaling pathways is avoided

Signal

molecule Plasma

membrane

Receptor

Scaffolding protein

Three different protein kinases

Scaffolding proteins are large relay proteins to which other relay proteins are attached

Scaffolding proteins can increase the signal transduction efficiency

Some receptors and signal transduction protein are localized

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

Clustering of membrane proteins mediated by adapter domains

Protein motifes

(1) Mediate protein-protein interactions (2) Determine the location of signaling proteins

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

SH2 PH

signal

receptor

SH3 PPP

Lipid eg PIP3

P P YP

Tyr K

effects

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

Amplifier proteins: usually either enzymes or ion channels that enhance the signal they receive

Transducer proteins: convert the signal to a different form e.g. adenyl cyclase Bifurcation (分枝)proteins: spread the signal from one signaling pathway to another

Different kinds of intracellular signaling proteins along a signaling pathway from the cell surface to the nucleus

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)

Tyrosine kinase receptors

– 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.

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

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.

• 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.

Tyrosine Kinase Receptors

Note steps involved:

1. Ligand Reception 2. Receptor Dimerization 3. Catalysis (Phosphorylization) 4. Subsequent Protein Activation 5. Further Transduction

6. Response

Tyrosine Kinase Receptors

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.

潛在

TGFR: receptor serine/

threonine kinase Smad: transcription factor TFE: transcription factor

phosphorylation

TGF- receptor and the direct activation of Smads

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 Complexes with other transcriptional

factor = Smad4 Complex moves in nucleus Activates gene

Gene = plasminogen activator inhibitor  no cell growth

kidney intestinal epithelial cells Membrane Form of Guanylyl Cyclase

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 cGMP- dependent protein kinase (G-

Kinase) GTP ^cGMP

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

The hormone-receptor complex has an exposed DNA binding siteand can activate transcription directly (or, more typically as a homo- or hetero-dimer) This usually initiates a cascade of

transcription events

Intracellular receptors (nucleus receptor)

Nuclear 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):

Ligand binding of the receptors triggers the formation of a dimeric complexthat 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 daysafter plasma concentration is zero.

response

Some signaling molecules that bind to intracellular receptors

cortisol

thyroxine

estradiol testosterone

Vitamin D3

Retinoic acid 一種腎上腺

皮質內泌素

雌二酮 睪固酮

甲狀腺素

維他命Ａ酸

Nuclear Receptors

• 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)

The nuclear receptor superfamily

Hinge 鉸縺

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)

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

Nuclear receptor family (steroid)

Early primary response (A) and delayed secondary response (B) that result from the activation

of an intracellular receptor protein

Ligand gate Ion-channel receptors

Ligand binding changes the conformation of the

receptor so that specific ions flow through it

Types of Membrane Ionic Channels

Non-gated channels: leakage channels open at rest

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

Nongated

ion channels and the resting membrane potential

Ion Channel (non-gate)

Generation of electrochemical gradient across plasma membrane 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 channel characterizations multi-subunit, transmembrane protein complexes

complex is both the receptor andion channel stimuli: chemical, stretch, voltage or light

stimulus induces conformational change to open orclose ion channel

 Light gated channels respond to light; in the eyes

 Mechanically gated channels respond to vibration or pressure - in the ear, touch

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⁺

NMDA receptor-Ligand gated channel

Ion-channel-linked receptors

Convert chemical signals ==> electrical signals

Extracellular ligand-gated

nicotinic ACh (muscle): 

 (embryonic), 

 (adult) nicotinic ACh (neuronal): (2-10), (2-4)

glutamate: NMDA, kainate, AMPA P2X (ATP)

5-HT

GABA

: (1-6), (1-4),  (1-4), , , (1-3) Glycine

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

• Intracellular ligand-gated channels:

• Ca

controlled K

channel

• ATP-sensitive K

channel

• IP

-operated Ca

channel (in the ER

membrane)

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 Non-Gated

Na

-channel - voltage Na

-channel Na

-channel - chemical

K

-channel - voltage K

-channel K

-channel - chemical

Cl

-channel - chemical Cl

-channel

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 C

-gated Ca

ryanodine receptor Ca

IP

-gated Ca

IP

-gated Ca

Ca

-gated K

Ca

-gated non-

selective cation

Ca

-gated Cl

cAMP cation cGMP cation cAMP chloride ATP Cl

volume-regulated Cl

arachidonic acid-

activated 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 (GABA_B)

• 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