A2 Trasduzione Del Segnale 03 10 2014.Ppt

8/18/2019 A2 Trasduzione Del Segnale 03 10 2014.Ppt

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Receptors in the Plasma Membrane• Most water-soluble signal molecules (eg. epinephrine, insulin, growth hormones)

bind to specific sites on receptor proteins in the plasma membrane

•

There are three main types of membrane receptors:

Ion channel-linkedreceptors

G-protein-linkedreceptors

Enzyme-linkedreceptors


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Ion Channel-linked Receptorssuch as receptor for glutamate, serotonin and acetylcholine

act as a gate when the receptor changeshape

are involved in rapid synaptic signalingbetween electrically excitable cells.

usually associated with a change in thecell’s membrane potential.

This type of signaling is mediated by asmall number of neurotransmitters thattransiently open or close the ion channelformed by the protein to which they bind.


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Receptors with intrinsic (a) or associated (b)enzymatic activity:

Receptor tyrosine kinases (RTKs) areprotein kinases that phosphorylates

tyrosine groups.

typically influencecell proliferation and differentiation.

Receptors that Interact withCytoplasmic JAK Kinases: Just

Another Kinase

such as cytokines receptorLigand molecule binds to the receptorand causes a conformational changewithin the receptor that leads toactivation of the JAK kinase.


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Extracellular region variable, with many different motifs

Usually cross membrane only once by a single transmembrane alpha-helix

Intracellular region contains conserved catalytic domains

Receptor tyrosine kinases (RTKs)The most wide family ofRTKs: Ephineprins can actsimultaneously as ligandsand receptors


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General structure and ligand-induced activation of receptor tyrosine kinases(RTKs)

Ligand promotes formation of RTK dimers, by different mechanisms:

Ligand itself is a dimer (PDGF) One ligand binds both monomers (GH)

Dimerization allows trans-phosphorylation of catalytic domains, whichinduces activation of catalytic (Y-kinase) activity

Activated TK domains phosphorylate each other and proteins nearby,sometimes on multiple tyrosines

Y~P residues recruit other signaling proteins, generate multiple signals


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How does dimerization activate RTKs?Growth Factor Receptors (like many kinases) have sites in their T loops at which

phosphorylation activates

Dimerization induces T-loopphosphorylation in trans

Phosphorylation of Y (one or more)in T-loop causes it to move out ofthe way of the active site.

Once activated, each monomer can phosphorylate nearby Y residues inthe other, as well as in other proteins

T-loopCat. loop

Y1162 occupies theactive site

Substrate Ysits in active site

Y1162flips out


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Recruitment of signal-transduction proteins to the cell membrane by binding tophosphotyrosine residues in activated receptors.

SH2 and PTB Domains Bind to SpecificSequences Surrounding PhosphotyrosineResidues


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G Protein–Coupled Receptors (GPCRs)

Receptor is associated with a groupof heterotrimeric proteins known asG proteins.

The G proteins can activate, orinactivate, plasma membrane effector

proteins that function as ion channelsor enzymes

and g subunits have covalently attached lipid anchors that bind a G-protein to theplasma membrane cytosolic surface.

The heterotrimeric G proteins contains 3subunits

The subunit (G

) binds GTP, and canhydrolyze it to GDP + Pi.

The subunit binds receptor, and forms astable complex with g subunit


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11Schematic diagram of the general structure of G protein–coupled receptors.

GPCR family


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Figure 15-31 Molecular Biology of the Cell (© Garland Science 2008)

Heterotrimeric G-protein


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Switching mechanism for monomeric and trimeric G proteins.


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Model of Ligand induced Activation of effector proteins associated with GPCRs


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G Protein–Coupled Receptors (GPCR) family

Human genome encodes for27 Ga 5 Gb

13 Gg

different G g display same functionG are divided in subclassis


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G Protein–Coupled Receptorsthat activate or inhibit

Adenylyl Cyclase

G Protein–Coupled Receptorsthat activatePhospholipase C

GPCR family include

G Protein–Coupled Receptorsthat activate

cGMP phosphodiesterase


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Ligand binding to Gs-coupled receptors causes activation of adenylyl cyclase, whereas ligand binding to Gi-coupled receptors causes inhibitionof the enzyme. The Gbg subunit in both stimulatory and inhibitory G proteins is

identical; the Ga subunits and their corresponding receptors differ. Ligand-stimulated formation of active Ga·GTPcomplexes occurs by the same mechanism in both Gs and Gi proteins. However, Gs·GTP and Gi·GTP interactdifferently with adenylyl cyclase, so that one stimulates and the other inhibits its catalytic activity. [See A. G.Gilman,1984, Cell 36:577.]

Hormone-induced activation and inhibition of adenylyl cyclase by different GPCRs in adipose cells.

G Protein–Coupled Receptors That Activate or Inhibit Adenylyl Cyclase


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! Cholera toxin catalyzes covalent modification of Gs in intestinal epithelial cells

• ADP-ribose is transferred from NAD+ to an arginine residue at the GTPaseactive 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 acysteine residue of the inhibitory Gi , making it incapable of exchanging GDP forGTP in respiratory epithelial cells

• The inhibitory pathway is blocked.

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


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CH2

HHOH OH

H HOOP

O

HHOH OH

H HO

CH2

N

N

N

NH2

OP

O

O

NO

(CH2)3

NH

C NH2+

protein

NH

O

H

CNH2

O

CH2

H

N

HOH OH

H HOOP

O

HHOH OH

H HO

CH2

N

N

N

NH2

OP

O

O

O

NO

H

CNH2

O

NH

+

+

(CH2)3NH

C NH2+

protein

NH2

NAD+

nicotinamideArg

residue

ADP-ribosylated protein

(nicotinamide

adenine

dinucleotide)

ADP

ribosylation


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1st messenger binds to GPCR receptor(a hormone such as epinephrine)

Receptor activates Gs protein

Gs protein activates adenylyl cyclase

Schematic diagram ofmammalian adenylyl cyclases.

3D crystal structureof Gs·GTPcomplexed withtwo fragmentsencompassing thecatalytic domain ofadenylyl cyclase

G Protein–Coupled Receptors that activate adenylyl cyclase


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Adenylyl cyclase, activated by GPCRsin response to an extracellular signal,

converts a molecule of ATP into cyclic AMP (cAMP), a “second messenger ”.

cAMP then attaches to and activatescAMP-dependent protein kinases thatcan phosphorylate and activate enzymesused in cellular responses.

The phosphodiesterase enzymes“terminate” the second messenger cAMP.

Caffeine and theophylline, the activeing red ien t s o f co f f ee and t ea

respectively, inhibit phosphodiesteraseactivity


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cAMP-induced activation of protein kinase A (PKA). [movie]

At low concentrations of cyclic AMP (cAMP), the PKA is an inactive tetramer. Binding of cAMP to the regulatory(R) subunits causes a conformational change in these subunits that permits release of the active, monomericcatalytic (C) subunits. (b) Cyclic AMP is a derivative of adenosine monophosphate. This intracellular signalingmolecule, whose concentration rises in response to various extracellular signals, can modulate the activity ofmany proteins.


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Molecular basis of the allosteric regulation of the regulative subunits of pKA.

pKA contains two regolativesubunits interconnected by a

dimerization domainEach R subunit contains two sitesfor cAMP (CNB-A and CNB-B).Binding of cAMP to each Rsubunit occurs in a cooperativemanner: binding of cAMP to CNB-B decrease the kd of cAMP for

CNB-A.When cAMP is bound to CNB-Athe R subunit undergoes toconformational changes thatdisplaces the associated C-subunit


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Figure 15-36 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)


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Figure 15-36 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)


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Amplification of an external signal downstream from a cell-surface receptor.

The cAMP system rapidly amplifies the responsecapacity of cells: here, one “first messenger” ledto the formation of one million product molecules.


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Cells can respond via the cAMP pathways using a diversity of cAMP-dependent

enzymes, channels, organelles, contractile filaments, ion pumps, and changes ingene expression.


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G Protein–Coupled Receptors that activate Phospholipase C

1st messenger binds to GPCR receptor

Receptor activates phospholipase C

Phospholipase C produces the secondary

messengers diacylglycerol (DAG), and

inositol triphosphate (IP3)


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Synthesis of DAG and IP3 from membrane-bound phosphatidylinositol (PI).


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33Inositol 1,4,5-Trisphosphate (IP3) Triggers Release of Ca2+ from the Endoplasmic ReticulumDiacylglycerol activates PKC

Signal transduction pathway downstream G Protein–Coupled Receptors Activating Phospholipase C


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Figure 15-41b Molecular Biology of the Cell (© Garland Science 2008)

Ca2+ as second messenger in signal trasduction


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Oscillation of [Ca2+] in the citosol of a liver cell affects cell response

The frequency ofCa2+ spikes reflectsthe potency of asignal

In the pituitary cellsto each Ca2+ spikecorresponds a fasthormone secretion.

In other cells eachspecific frequency of

Ca2+ spikesinduces a specificpattern of genesactivation


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Calmodulin, a Ca2+ binding protein that regulate the activity of several kinases andother enzymes

Ca2+ can make calmodulin ableto bind to a target protein

Two or more Ca2+ have to bind calmodulin for its activation


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Activation of gene expressionfollowing ligand binding to Gsprotein–coupledreceptors

Activation of the Tubby transcriptionfactor following ligand binding toreceptors coupled to Go orGq


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G Protein–Coupled Receptors that activate cGMP phosphodiesterase: Rhodopsin

Photon isomerize 11-cis-retinal molecule

Large Rhodopsin molecule change conformation and activate the associatedG-proteins, trasducins

Trasducins activate cGMP phosphodiesterases

cGMP phosphodiesterases hydrolyzes cGMPs

cGMPs reduction closes cGMP-dependent Na channel that prevent ions-dependent membrane hyperpolarization

Alteration of the membrane potential and reduction in neurotrasmitter release


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A2 Trasduzione Del Segnale 03 10 2014.Ppt

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