Lecture 15: Intracellular Signaling

Give a brief overview of G-protein activation.

• inactive: G-alpha-beta-gamma (with G-alpha-GDP) bound to G-beta-gamma

• GPCR activation induces an exchange of GTP for GDP

• G-alpha-GTP dissociates from G-beta-gamma

• active: G-alpha-GTP and G-beta-gamma influence effector proteins

Give a brief overview of G-protein inactivation.

• G-alpha inactivates by slowly converting GTP to GDP (by its intrinsic GTPase activity)

• RGS (regulator of G-protein signaling) proteins accelerate the rate of GTP hydrolysis by G-alpha

• G-beta-gamma recombine with G-alpha-GDP and becomes inactive

How can a neurotransmitter produce a specific response if G-protein coupling has the potential for such a diversity of effector?

• a given neuron has only a subset of receptors, G proteins, and effectors, thereby limiting possible signaling pathways (ie. transducin the visual system)

• spatial compartmentalization restricts a given colocalized receptor to a limited set of effectors in the same location (ie. localized only to nerve terminals or PSD)

• selective affinities between cognate sets of receptors, G proteins, and effectors exist (specific macromolecular complex)

• diverse regulators of G protein signaling (RGS) to promote GTP hydrolysis and terminate specific G-alpha-GTP

True or False: The retina contains millions of light-sensitive cells (rods and cones) that receive and organize visual information.

true

List the pathway that leads to activation of transducin.

1. light modifies and activates rhodopsin—leading to activation of transducin (G_t)

2. a single photon activates 500 transducin (G_t-protein)

3. a transducin activates 500 PDE, each of which hydrolyzes 100 cGMP

4. cGMP-dependent cation channels close, resulting in membrane hyperpolarization

True or False: Retinal is a polyene chromophore.

true

What is adenylate cyclase?

a 120 kD protein that produces cAMP from ATP

What stimulates adenylate cyclase?

G-s

What inhibits adenylate cyclase?

• G-i

• G-o

• G-z

What degrades cAMP?

cAMP phosphodiesterase (PDEs)

What are some examples of ligands that involve adenylate cyclase?

• dopamine

• NE

• E

• 5HT

• histamine

• neuropeptides like VIP and somatostatin

What does phospholipase CB do?

hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) to diacylglycerol (DAG) and inositol 1,4,5-triphsophate (IP₃)

What stimulates phospholipase CB?

• pertussis toxin-sensitive G-q

• insensitive G-i

What are some examples of ligands involved with phospholipase CB?

• ACh (M1 and M3)

• NE

• glutamate

• 5HT

• neuropeptides such as neurotensin and substance P

What does RGS6 do in cerebellar granule neurons?

• limits GABA_B receptor-mediated GIRK channel activation

• limits serotonin receptor (5-HT1AR)-mediated inhibition of adenylate cyclase (AC) and anti-depressant actions

True or False: There is integration of cellular stimuli to the phosphorylation state of proteins.

true

True or False: Phosphorylation and dephosphorylation alter functions of the proteins over broad time (ms - hr).

true

True or False: Distinct signaling pathways can converge on to the same or different substrates.

true

What are some functional states that phosphorylation and dephosphorylation regulate?

• increase/decrease catalytic activity of an enzyme

• alter conductance and gating of an ion channel

• desensitization of a receptor

• induce protein conformational change

• alter interaction with other proteins

What is PKA?

• cAMP-dependent protein kinase

• a Ser/Thr kinase that regulates many neuronal processes including gene expression, neurotransmitter synthesis, MAP2, ion channels, and glutamate receptors

Briefly describe the classical activation of PKA.

cAMP binds to PKA regulatory subunit R1, which then releases catalytically active subunit

What does preferential degradation of inhibitory R1 subunit of PKA lead to? What does this lead to?

persistent activity of PKA in the absence of cAMP—leading to cognitive kinase

What is PKC?

• protein kinase C

• a Ser/Thr kinase that regulates many cellular processes such as exo-, endo-cytosis, plasticity, gene expression, cell cycle, glutamate receptors, and ion channels

Briefly describe the classical activation of PKC.

DAG increases the affinity of cPKC inhibitory domain for Ca2+ affinity of and phosphatidylserine and induces its translocation to the plasma membrane and activation of cPKC

What does the proteolytic removal of cPKC inhibitory domain produce? What does this lead to?

it produces a constitutively active kinase called protein kinase M (PKM)—leading to cognitive kinase

What is CAMK?

• Ca2+-CaM dependent kinase

• a Ser/Thr kinase with broad substrates such as glutamate receptors, tyrosine hydroxylase, Ca2+ channels, MAP2, and transcription factors

Where is CAMK present?

• in every tissue

• enriched in neurons

What are the domains on CAMK?

• catalytic

• autoinhibitory (inhibitory and calmodulin binding)

• self-association (variable inserts)

Briefly describe the classical activation of CAMK.

Ca2+-bound CaM binds to autoinhibitory domain of CaMKII and exposes catalytic domain

What produces persistently active CaMKII?

• binding of the NMDA receptor subunit GluN2B C-termini

• autophosphorylation of CaMKII at Thr-286

What does a persistently active CaMKII lead to?

cognitive kinase

What do Tyr kinases play a role in?

• synaptic transmission

• multiple ion channels

• neuronal signaling and development

What does receptor protein tyrosine kinases mediate?

cellular processes:

• growth

• motility

• differentiation

• metabolism

• synapse

What does ligand binding and dimerization of the receptor protein tyrosine kinase lead to?

conformational changes and trans-autophosphorylation of each tyrosine kinase domain (TKD) and release of the cis-autoinhibition

What do protein phosphatases do?

catalyze the hydrolysis of the ester bond of the phosphorylated amino acids

Which processes are protein phosphatases critical for?

• synaptic transmission

• neuronal development

• neuronal signaling

• neural plasticity

What does activation of CNA (calcineurin) require?

Ca2+ and CaM binding

True or False: There is cross talk between kinases and phosphatases.

true

What activates and inhibits DARPP-32?

• PKA phosphorylates and activates

• PP2B dephosphorylates and inhibits

What does PTP stand for?

protein tyrosine phosphatase

True or False: Neuronal activity regulates transcription via diverse signal pathways and activation of protein kinases and phosphatases.

true

True or False: Gene transcription is important for neuronal plasticity, survival, and signaling.

true