3-2) Lecture 22 - Signal Transduction II

What are the key challenges in signal transduction?

Specificity,

Transmission,

Amplification,

Termination/desensitization

Where do many of the important signals come from?

The original cue is a change in the environmental condition, often in the form of a chemical molecule.

Most extracellular signaling molecules are ___ and cannot readily cross ___ ___

polar, hydrophobic membrane

What are potential signals from outside of the cell?

Soluble signaling molecules in extracellular fluids,

molecules present on the surface of adjacent cells,

signals from extracellular matrix,

physical signals

What are some soluble signaling molecules in extracellular fluids?

Hormones, Cytokines (immune response), Mitogens (cell proliferation)

What are some physical signals?

Electrical signals (ions) and sensory signals (light, pressure, odors)

What are the general strategies to transfer signals across the membrane?

Signaling molecules bind to transmembrane receptors, leading to activation of intracellular enzymes

Control the opening of membrane ion channels

passively diffuse across the membrane into the cytosol.

What are examples of how signaling molecules bind to transmembrane receptors, leading to activation of intracellular enzymes?

G-protein coupled receptors (indirectly), and receptor enzyme (directly)

What are examples of signaling molecules controlling the opening of membrane ion channels?

Gated ion channels open and close in response to membrane potential, ion concentrations, and metabolites.

What are examples of signaling molecules passively diffusing across the membrane into the cytosol?

Steroid hormones (nuclear receptors), which are neutral

Gas molecules (NO, O2). For these cases, the receptors are in the cells.

Ligand-receptor interaction is of __ __ and __ __

high affinity, high specificity

What are the three pieces of a ligand-receptor interaction?

Extracellular domain that recognizes and binds external ligands,

a transmembrane domain that consists of one more more transmembrane helices,

an intracellular domain interacts with the interior of the cell that relays the signal.

Signaling molecules must bind to its cognate receptor with __ __ but not to _ ___ ___ ___

high affinity / other structurally similar ones.

Why is it difficult for signaling molecules to bind to its cognate receptor?

Concentration of signaling molecules in the environment and number of receptors in the membrane are often very low.

Competing signaling molecules and particularly receptors are often structurally similar.

What is an effective strategy to modulate biological signaling?

Targeting ligand-receptor interaction.

Agonist

mimic natural ligands and cause activation of the receptor

Antagonist

bind to the receptor but do not activate the receptor

G protein-coupled receptors (GPCRs)

receptors that act through a member of the heterotrimeric G protein family.

How is GPCR signal transduction related in biological functions of eukaryotes?

Mediate signaling in vision, smelling, regulation of blood glucose level, regulation of blood pressure, among others.

Ligands include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters.

What are two important GPCR signals for metabolism?

glucagon, epinephrine

What are the three essential components of signal transduction through GPCRs?

A plasma membrane receptor ;

a heterotrimeric G protein that cycles between active (GTP-bound) and inactive (GDP-bound) forms ;

an effector enzyme (or ion channel) in the plasma membrane that is regulated by the activated G protein.

What is the most common type of signal transduction mechanism found in eukaryotes?

Signal transduction through GPCRs.

Ligand binding induces GPCR…

conformational change.

Seven-transmembrane (7-TM) receptors

found in GPCRs.

Ligand binding to the extracellular domain of the GPCR induces…

concerted conformational change on the intracellular surface of the receptor that involves alterations in the relative packing of the seven transmembrane helices.

How does the concerted conformational change on the intracellular surface of a GPCR impact GPCR?

These changes increase the affinity of a GPCR for its cognate G protein.

Activated GPCR acts as a ___ for heterotrimeric G protein

GEF

How does GPCR act as a GEF?

Activated GPCR associates with a GDP-bound heterotrimeric G protein and acts as a GEF to catalyze GDP/GTP for the Gα subunit.

In GPCR, how does GTP binding affect the Gα subunit?

GTP-binding induces conformational change in the Gα subunit, leading to its dissociation from both the receptor and from the Gβγ dimer.

In GPCR, when activated, both separated Gα subunit and Gβγ dimer can…

bind to downstream effector enzymes (or ion channels) to continue signal transmission.

Heterotrimeric G proteins

Have α, β, and γ subunits(~16 α subunits in humans.)

Gα subunits of heterotrimeric G proteins

Gsα - stimulates adenylate cyclase (“s” for stimulation),

Giα inhibits adenylate cyclase (“i” for inhibition),

Gqα activates phospholipase C.

G12/13α activates RhoGEF (a GEF for small G protein Rho).

How does a weak input signal lead to a robust response?

During signal transduction, signals are amplified when mediated by an enzymatic reaction. '

When an enzyme activates another enzyme, the number of affected molecules increases geometrically.

Signals are generally NOT amplified when mediated by a binding interaction.

PIP3 signaling pathway

PIP3 recruits PH domain containing PDK1 protein kinase to the membrane. PIP3 also recruits PH domain containing Akt protein kinase (PKB), a substrate of PDK1, to the membrane.

Phosphorylation of Akt by PDK1 activates Akt. Activated Akt moves through the cell to phosphorylate targets to promote.

How do cholera toxin and pertussis toxin interfere with GPCR signaling?

Cholera toxin causes ADP-ribosylation of Gsα, making the modified Gsα stabilized in the GTP form and continuously activating PKA, which leads to excessive loss of NaCl and large amounts of water into the intestine.

Pertussis toxin causes ADP ribosylation of Giα, preventing Giα from interaction to its receptor, trapping it in the “off” state.

What is the difference between termination and desensitization of signals?

Termination is the overall shutting down or turning off of a signaling pathway, while desensitization damps cell’s response when the signal persists and represents adaptation to the changed environment.

What are mechanisms that cause termination of GPCR signaling?

Ligand dissociation (due to concentration drops below the Kd for its receptor),

GTP hydrolysis catalyzed by GAP (GTPase which is stimulated by GTPase activator proteins [GAPs] activity of G protein hydrolyzes GTP bound to the Gα subunit),

cAMP hydrolysis catalyzed by PDE (turns back to AMP by cyclic nucleotide phosphodiesterase, second messenger degraded),

dephosphorylation catalyzed by phosphatase (hydrolyze phosphorylated amino acids).

What are some examples of desensitization of GPCR signaling?

The activated receptor is phosphorylated by G Protein-Coupled Receptor kinases (GRKs). Phosphorylated receptor is bound by arrestin which prevents Gα from binding. The arrestin-receptor complex is removed by the plasma membrane.

Receptor Enzymes

consists of an extracellular ligand-binding domain, a single transmembrane helix (with exceptions), and an intracellular domain with enzyme activity.

Receptor tyrosine kinase (RTK)

tyrosine protein kinase domain. Binds peptide ligands (e.g. insulin and growth factors) that regulate proliferation, growth, differentiation, or migration.

What is the most common intracellular enzyme domain?

RTK

How does ligand binding lead to activation of receptor tyrosine kinase?

Ligand binding induces receptor dimerization.

This can happen through many mechanisms:

• Ligand itself may be a dimer,

• Ligand has two distinct receptor bindings sites,

• Ligands are membrane bound and are physically clustered on the cell surface, or

• Ligand does not directly mediate dimerization but binding induces conformational change that increases the affinity between receptors.

Why does a RTK dimerize after ligand binding?

This is because for a receptor with a single transmembrane helix, conformational change in the extracellular domain cannot be easily transmitted.

What does dimerization of RTKs lead to?

Trans-autophosphorylation, which activates the receptor enzyme.

How does protein dimerization work after ligand binding?

Dimerization brings two intracellular kinase domains close to each other.

The catalytic domains phosphorylate each other on the activation loop.

The kinase activity increases, and more tyrosine residues on the C-termini become phosphorylated.

Phosphorylated tyrosine residues create binding sites for downstream effector proteins.

Activated RTKs function as nucleation sites for…

protein complexes that recruits key signaling molecules to membrane vicinity.

Activated receptors promote…

the formation of signaling complexes

Phosphorylated tyrosine on activated RTKs can function as docking sites for proteins containing…

modular p-Tyr binding domains such as SH2.

RTKs can phosphorylate…

adaptor/scaffold proteins which can function as docking sites for additional proteins.

Mitogen signaling pathway begins with…

the activation of Ras

The intracellular signaling pathway for several mitogens begins with…

the docking of adaptor protein Grb2 to the phosphorylated receptor.

What does Grb2 contain?

An SH2 domain and two SH3 domains.

What do the SH2 and SH3 domains of Grb2 do?

SH2 domain binds to phospho-tyrosine residues on the receptor.

SH3 domains bind to proline-rich sequences in signaling protein Sos, a GEF for small G protein Ras.

What does Sos do when it is recruited to the membrane vicinity?

It catalyzes GDP/GTP exchange, activating Ras.

Mitogens

extracellular signaling molecules that induce mitosis and cell division: Epidermal growth factor (EGF), Fibroblast growth factor (FGF), Platelet-derived growth factor (PDGF)

Activated Ras initiates…

MAP kinase pathway

What are the molecular events of the MAP Kinase pathway?

When bound to GTP, Ras can activate Raf kinase.

Raf kinase phosphorylates and activates MEK kinase.

MEK kinase phosphorylates and activates Erk kinase.

Phosphorylated Erk enters the nucleus and phosphorylates transcription factors to activate gene transcription, producing changes in the cell

What are the proteins involved in the MAP kinase cascade?

Erk is the MAPK (mitogen-activated protein kinase) family;

MEK is the MAPKK family, which activates the MAPK;

Raf is the MAPKKK family, which activates the MAP kinase kinase.

What is the Raf-MEK-Erk module used for?

To mediate cell responses to mitogenic signals.

The MAP kinase pathway is…

a signaling module in eukaryotes

What’s the general order of protein families for the MAP kinase pathway?

MAPKKK → MAPKK → MAPK

What are the molecular events downstream of receptor tyrosine kinase activation?

Dimerization mediated trans-autophosphorylation,

activated receptors promote the formation of signaling complexes,

mitogen signaling pathway begins with the activation of Ras,

activated Ras initiates MAP kinase pathway.

Adenylyl cyclase

a large integral membrane enzyme that catalyzes the synthesis of cAMP from ATP. it’s the effector enzyme in Gsα and Giα signaling

How is adenylyl cyclase activated and inhibited?

Activated by Gsα-GTP binding and inhibited by Giα-GTP binding. 

List an example of molecules that interact with the Gα subunit

Epinephrine binding to β-adrenergic receptor activates Gsα leading to an increase in cAMP concentration.

Dopamine binding to dopamine (D2) receptor activates Giα, leading to a decrease in cAMP concentration. 

Protein kinase A

A Ser/Thr kinase involved in regulating many functions in metabolism. Allosterically activated by cAMP.

PKA Holoenzyme

exists as a R2C2 tetramer that contains two catalytic subunits and two regulatory subunits.

The R subunit suppresses the kinase activity of the C subunit.

When cAMP binds to the R unit, the R subunit is released from the C subunit.

Free C subunit is catalytically active.

Phospholipase C (PLC)

the effector enzyme in Gqα signaling. Catalyzes the synthesis of DAG and IP3 from PIP2.

IP3 causes the influx of Ca2+ from the ___ into the ___

ER, cytosol

Ca2+ activates numerous cytosolic kinases, in some cases by acting through __

calmodulin

Ca2+ and DAG are required for…

Protein kinase C (PKC) activation.

Give an example of a molecule that interacts with the Gqα signaling.

Glutamate binding to glutamate receptor (mGluR) activates Gqα, leading to calcium signaling and activation of PKC.

Ras

a family of small GTPase proteins that act as binary molecular switches inside cells, regulating essential cellular processes like growth, division, and survival. On = bound to GTP, off = bound to GDP.

Raf (MAP3K)

activates MEK, the MAPKK (step one)

MEK (MAP2K)

activates Erk, the MAPK (step 2)

Erk (MAP Kinase)

when phosphorylated, can enter the nucleus and phosphorylate transcription factors to activate gene transcription, producing changes in the cell, such as cell division.

Insulin receptor

an α2β2 tetramer that consists of two identical sets of subunits (α and β) linked by disulfide bonds.

Insulin binding does not induce protein dimerization, but instead…

trigger conformational changes in the extracellular changes in the extracellular α subunits.

The conformational changes bring the intracellular kinase domains of β subunits closer together for trans-autophosphorylation.

When the insulin receptor is auto-phosphorylated, what is one of its targets?

A scaffold protein IRS1 (insulin receptor substrate 1).

IRS1 binds to the autophosphorylated insulin receptor via its PTB domain (another pTyr binding domain) and to the membrane via its PH domain.

What are the two major outcomes of insulin action?

Regulation of gene expression to promote cell growth (mitogenic) and regulation of glucose homeostasis (metabolic)

How is the mitogenic axis mediated?

By Grb2 and Shp2 (an SH2-containing protein phosphatase) through the Ras-MAPK pathway.

Insulin signaling can also lead to…

the activation of PI3K

Regulation of glucose homeostasis by insulin is mediated through…

phosphoinositide 3-kinase (PI3K) pathway.

Phosphoinositide 3-kinase (PI3K) pathway

phosphorylated IRS-1 provides docking sites for the SH2 domain within the PI3K regulatory subunit p85. Through its interaction with p85, the catalytic subunit of PI3K, p110, is brought to the membrane. PI3K catalyzes the conversion of PIP2 to PIP3.

Once the IRS is phosphorylated on several of its tyrosine residues by the receptor…

it becomes the point of nucleation for proteins that carry the message from the receptor to intracellular targets.

PIP3 activates…

additional protein kinases

PIP3 recruits…

PH domain containing PDK1 protein kinase to the membrane. It also recruits a PH domain containing Akt protein kinase (PKB), a substrate of PDK1, to the membrane.

PDK1 Kinase

contains a PH domain, recruited by PIP3 to the membrane. Phosphorylates Akt protein kinase to activate Akt.

Akt protein kinase (PKB)

phosphorylated by PDK1 to activate Akt.

What does activated Akt do?

It moves through the cell to phosphorylate targets to promote glucose uptake from blood stream (muscle and fat) and glycogen synthesis (liver and muscle).