Cellular Signal Transduction

Organisms can function normally only if: All the cells of their different organs communicating

effectively with their surroundings. Once a cell picks up a hormonal or sensory signal, it

must transmit this information from the surface to the interior parts of the cell—Example, to the nucleus.

This occurs via signal transduction pathways that are: very specific, both in their activation and in their downstream actions.

Thus, the various organs in the body respond in an appropriate manner and only to relevant signals.

Signal Transduction The transmission of molecular signals from a cell's exterior to its interior.Molecular signals transmitted between cells by:1. Hormones 2. Other chemical factors3. Sensory signals received from

the environment, in form of: Light, Taste, Sound, Smell, and Touch.

Upon Environment changes:

Monad: responds directly

Multicellular organisms:

Respond with signal through a system of intercellular or intracellular communication， and consequently regulate functions of organisms.

Signaltransduction

Signaling molecule

Receptor of target cell

Intracellular molecule

biological effect

1. Signaling Molecules

Signaling molecules

• Released by signal-producing cells,• Transfer biological signals to their

target cells to initiate specific cellular responses.

Two types..

• 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

(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 lipid derivatives

• IP3 carbohydrate

derivatives

• cAMP cGMP nucleotides

• Ras, JAK, Raf proteins

Second messenger:

Small molecules synthesized in cells in response to an external signal, responsible for intracellular signal transduction.

Such as Ca2+, DG, Cer, IP3, cAMP, cGMP

Third messengers:

The molecules which transmit message from outside to inside of nucleous or from inside to outside of nucleous, also called DNA binding protein.

Effect by membrane receptors

Effect by intracellular receptors

Intracellular molecules

Extracellular molecules

Signal molecules

cAMP, cGMP, IP3, DG, Ca2+

Proteins and peptides:

Hormones, cytokines

Amino acid derivatives:

Catecholamines

Fatty acid derivatives:

Prostaglandins

Steroid hormones, Thyroxine, VD3

2. Receptors

Receptors

Specific membrane proteins, 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;

Example:

A hormone is the ligand for its specific

protein receptor.

• Membrane receptors

membrane

Glycoprotein

• Intracellular receptors

Cytosol or nuclei

DNA binding protein

(1) Ligand-gate ion channels type (cyclic receptor)

ligand→receptor→ion channel open or close

1. membrane receptors

Properties of binding of H and R

• highly specificity

• highly affinity

• saturation

• reversible binding

• special function model

Control of receptor activity

• Phosphorylation or dephosphorylation of R

• Phospholipid of membrane

• Enzyme catalyzed hydrolysis

• G protein regulation

(1) Recognize the special ligand

(2) Binding to special ligand

(3) Signal transduction biological effect

Function of receptor

Pathway of Signal Transduction

Many enzymes are regulated by covalent attachment of phosphate, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (serine, threonine, or tyrosine).

H3N+ C COO

CH OH

CH3

H

threonine (Thr)

H3N+ C COO

CH2

OH

H

serine (Ser)

A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein.

A protein phosphatase catalyzes removal of the Pi by hydrolysis.

P r o te in O H + A T P P r o te in O P

O

O

O

+ A D P

P i H 2 O

P r o te in K in a s e

P r o te in P h o s p h a ta s e

Phosphorylation may directly alter activity of an enzyme, e.g., by promoting a conformational change.

Alternatively, altered activity may result from binding another protein that specifically recognizes a phosphorylated domain. E.g., 14-3-3 proteins bind to domains that include

phosphorylated Ser or Thr in the sequence RXXX[pS/pT]XP, where X can be different amino acids.

Binding to 14-3-3 is a mechanism by which some proteins (e.g., transcription factors) may be retained in the cytosol, & prevented from entering the nucleus.

Protein kinases and phosphatases are themselves regulated by complex signal cascades. For example: Some protein kinases are activated by Ca++-

calmodulin. Protein Kinase A is activated by cyclic-AMP (cAMP).

P r o te in O H + A T P P r o te in O P

O

O

O

+ A D P

P i H 2 O

P r o te in K in a s e

P r o te in P h o s p h a ta s e

Adenylate Cyclase (Adenylyl Cyclase) catalyzes: ATP à cAMP + PPi

Binding of certain hormones (e.g., epinephrine) to the outer surface of a cell activates Adenylate Cyclase to form cAMP within the cell.

Cyclic AMP is thus considered to be a second messenger.

N

N N

N

NH2

O

OHO

HH

H

H2C

HO

PO

O-

1'

3'

5' 4'

2'

cAMP

Phosphodiesterase enzymes catalyze:

cAMP + H2O 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

OHO

HH

H

H2C

HO

PO

O-

1'

3'

5' 4'

2'

cAMP

Protein Kinase A (cAMP-Dependent Protein Kinase) transfers Pi from ATP to OH of a Ser or Thr in a particular 5-amino acid sequence.

Protein Kinase A in the resting state is a complex of: • 2 catalytic subunits (C) • 2 regulatory subunits (R).

R2C2

R2C2

Each regulatory subunit (R) of Protein Kinase A contains a pseudosubstrate sequence, like the substrate domain of a target protein but with Ala substituting for the Ser/Thr.

The pseudosubstrate domain of (R), which lacks a hydroxyl that can be phosphorylated, binds to the active site of (C), blocking its activity.

R2C2 + 4 cAMP R2cAMP4 + 2 C

When each (R) binds 2 cAMP, a conformational change causes (R) to release (C).

The catalytic subunits can then catalyze phosphorylation of Ser or Thr on target proteins.

PKIs, Protein Kinase Inhibitors, modulate activity of the catalytic subunits (C).

Pathway of G protein linked receptor

H R G protein Es

secondary messeger

Protein kinase

Phophorylation of Es or functional protein

Biological effect

Rhodopsin PDB 1F88

G Protein Signal Cascade

Most signal molecules targeted to a cell bind at the cell surface to receptors embedded in the plasma membrane. Only signal molecules able to cross the plasma membrane (e.g., steroid hormones) interact with intracellular receptors.

A large family of cell surface receptors have a common structural motif, 7 transmembrane a-helices.

Rhodopsin was the first of these to have its 7-helix structure confirmed by X-ray crystallography.

Rhodopsin is unique.

It senses light, via a bound chromophore, retinal.

Most 7-helix receptors have domains facing the extracellular side of the plasma membrane that recognize & bind signal molecules (ligands).

E.g., the b-adrenergic receptor is activated by epinephrine & norepinephrine.

Crystallization of this receptor was aided by genetically engineering insertion of the soluble enzyme lysozyme into a cytosolic loop between transmembrane a-helices.

-Adrenergic Receptor

PDB 2RH1

Lysozyme insert

ligand

The signal is usually passed from a 7-helix receptor to an intracellular G-protein.

Seven-helix receptors are thus called GPCR, or G-Protein-Coupled Receptors.

Approx. 800 different GPCRs are encoded in the human genome.

G-protein-Coupled Receptors may dimerize or form oligomeric complexes within the membrane.

Ligand binding may promote oligomerization, which may in turn affect activity of the receptor.

Various GPCR-interacting proteins (GIPs) modulate receptor function. Effects of GIPs may include: altered ligand affinity

receptor dimerization or oligomerization

control of receptor localization, including transfer to or removal from the plasma membrane

promoting close association with other signal proteins

G-proteins are heterotrimeric, with 3 subunits a, b, g.

A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein, designated Gs with alpha subunit Gsa.

Gs is activated, e.g., by receptors for the hormones epinephrine and glucagon.

The b-adrenergic receptor is the GPCR for epinephrine.

a & g subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface.

Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site.

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PP i

cytosol

GDP GTP

The asubunit of a G-protein (Ga) binds GTP, & can hydrolyze it to GDP + Pi.

The sequence of events by which a hormone activates cAMP signaling:

1. Initially Ga has bound GDP, and a,b, &g subunits are complexed together.

Gb,g, the complex of b & g subunits, inhibits Ga.

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PPi

cytosol

GDP GTP

2. Hormone binding, usually to an extracellular domain of a 7-helix receptor (GPCR), causes a conformational change in the receptor that is transmitted to a G-protein on the cytosolic side of the membrane. The nucleotide-binding site on Ga becomes more accessible to the cytosol, where [GTP] > [GDP]. Ga releases GDP & binds GTP (GDP-GTP exchange).

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PPi

cytosol

GDP GTP

3. Substitution of GTP for GDP causes another conformational change in Ga.

Ga-GTP dissociates from the inhibitory bg complex & can now bind to and activate Adenylate Cyclase.

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PP i

cytosol

GDP GTP

4. Adenylate Cyclase, activated by the stimulatory Ga-GTP, catalyzes synthesis of cAMP.

5. Protein Kinase A (cAMP Dependent Protein Kinase) catalyzes transfer of phosphate from ATP to serine or threonine residues of various cellular proteins, altering their activity.

AC

hormone signal outside GPCR plasma membrane

GTP GDP ATP cAMP + PPi

cytosol

GDP GTP

Turn off of the signal:

1. Ga hydrolyzes GTP to GDP + Pi. (GTPase).

The presence of GDP on Ga causes it to rebind to the inhibitory bgcomplex.

Adenylate Cyclase is no longer activated.

2. Phosphodiesterases catalyze hydrolysis of cAMP AMP.

3. Protein Phosphatase catalyzes removal by hydrolysis of phosphates that were attached to proteins via ProteinKinase A.

Signal amplification is an important feature of signal cascades:

One hormone molecule can lead to formation of many cAMP molecules.

Each catalytic subunit of Protein Kinase A catalyzes phosphorylation of many proteins during the life-time of the cAMP.

Different isoforms of Ga have different signal roles. E.g.:

• The stimulatory Gsa, when it binds GTP, activates Adenylate cyclase.

• An inhibitory Gia, when it binds GTP, inhibits Adenylate cyclase.

Different effectors & their receptors induce Gia to exchange GDP for GTP than those that activate Gsa.

The complex of G ,b g that is released when Ga binds GTP is itself an effector that binds to and activates or inhibits several other proteins.

E.g., G ,b g inhibits one of several isoforms of Adenylate Cyclase, contributing to rapid signal turnoff in cells that express that enzyme.

Cholera toxin catalyzes covalent modification of Gsa. • ADP-ribose is transferred from NAD+ to an arginine

residue at the GTPase active site of Gsa. • ADP-ribosylation prevents GTP hydrolysis by Gsa. • The stimulatory G-protein is permanently activated.

Pertussis toxin (whooping cough disease) catalyzes ADP-ribosylation at a cysteine residue of the inhibitory Gia, making it incapable of exchanging GDP for GTP. • The inhibitory pathway is blocked.

ADP-ribosylation is a general mechanism by which activity of many proteins is regulated, in eukaryotes (including mammals) as well as in prokaryotes.