BIPN140 Lecture 10: Intracellular Signaling

Su (FA16)

1. Overview of Chemical Signaling

2. G protein signaling & Phosphorylation

3. Nuclear Signaling

4. Receptor Tyrosine Kinase Signaling

Two Types of Neurotransmitter Receptors (Fig. 5.16)

(1) Both types of NT receptors can trigger intracellular signaling events (especially if the ligand-gated channel is permeable to Ca2+ or an VGCC can be activated subsequently).

(2) A neuron is also a cell => many types of signaling events take place in a neuron; some are mediated by neurotransmitter receptors and some are mediated by other types of receptors/signaling molecules.

Chemical Signaling in Neurons (Fig. 7.1)

Types of major intercellular chemical signaling: (1) synaptic; (2) paracrine (diffusion via interstitial fluid); (3) endocrine (via blood stream, throughout the whole body).

Three key components: chemical signal (signaling cells), receptor & target molecules (target cells)

Binding of the signaling molecules causes the conformation of the receptor to change.

Intracellular signal transduction: cellular events happen in the target cell upon receptor-ligand binding.

Advantages of Chemical Signaling (Fig. 7.1)

Versatility

Precise temporal control: from rapid (nAChR at NMJ, catching a ball) to long-lasting response (muscle metabolism, stress response, emotional state, etc). Having multiple levels of molecular interactions facilitates the intricate timing of these events to allow different types of integration.

Precise spatial control: between or within neurons; compartmentalization of signaling molecules allow precise spatial regulation (e.g. axon terminal v.s. cell body; dendritic spine v.s. shaft)

Signal amplification: one signaling molecule could result in activation of many effector enzymes or production of many 2nd messenger molecules.

Amplification in Signal Transduction Pathways (Fig. 7.2)

Activation of a single receptor can lead to activation of numerous G-proteins and effector enzymes. Each activated enzyme molecules generates a large number of 2nd messengers, which can then activate other downstream enzymes to catalyze reactions affecting a large number of target proteins.

Not every signaling step leads to amplification, but overall the cascade results in a tremendous increase in the potency of the initial signal.

Enzyme Enzyme

Depends on the half life of the activated receptor

Depends on the half life of the GTP-bound G

Three Classes of Cell Signaling Molecules (Fig. 7.3)

Four combinations:

1. Surface receptors activated by cell-impermeant ligands

2. Surface receptors activated by cell-attached or transmembrane ligands (e.g. ephrin/EphBreceptors), particularly important during development, requires direct contact between signaling and target cells.

3. Surface receptors activated by lipophilic ligands (CB1/endocannabinoids)

4. Intracellular receptors activated by cell-permeant agonists

Three classes of signaling molecules: secreted cell-impermeant, secreted cell-permeant (lipid soluble) & cell-associated (transmembrane) molecules.

Two major types of receptors: surface receptor and intracellular receptor.

Lipophilic (e.g. steroid hormone)

Categories of Cellular Receptors for Secreted Signals (Fig. 7.4)

For cell-impermeant molecules (water soluble or hydrophilic):

(1) ligand-gated ion channels

(2) enzyme-linked receptors

(3) GPCRs

Ligand binding domain (extracellular) & signaling domain (intracellular)

For cell-permeant molecules (lipid soluble or lipophilic or hydrophobic): intracellular receptors (in the cytosol, can translocate into the nucleus upon ligand binding, leading to transcription activation). For example, steroid hormones and their cytosolic receptors — binding of the ligand allows the receptor to enter the nucleus to trigger gene transcription.

e.g. membrane bound guanylyl cyclase

Types of GTP-binding Proteins: Large G Proteins (Fig. 7.5)

Heterotrimeric G proteins: (1) three subunits (, / complex) (2) subunit binds to GDP/GTP (intracellular GTP is abundant)(3) Agonist => GPCR =>GDP dissociates from the subunit, which binds to GTP => GTP

dissociates from and interact with target effectors (enzymes or ion channels). In some cases, can also activate the downstream effectors (e.g. mAChR => direct activation of K+ channels by , G protein-coupled inward rectifying potassium channels or GIRKs).

(4) Termination requires inactivation of the GPCR (phosphorylation/arrestin binding), and/or inactivation of the G protein (GTPase activating proteins, GAPs, such as RGS proteins –regulators of G protein signaling, or inactivation of the downstream effectors).

GEFs: Guanine nucleotide exchange factors

GAPs: GTPase activating proteins (the endogenous GTPase activity is usually too slow to terminate the signal)

Intracellular [GTP] is much higher than [GDP]. Therefore, as long as GDP is dissociated from the subunit, GTP can bind to the “free” subunit immediately.

GTP bound subunit has a reduced affinity to the activated GPCR => dissociation from the receptor.

G has endogenous GTPase activity => allowing turning off the response (but usually too slow, requires GAPs)

The length of G protein signal is usually controlled by the duration of the GTP-bound G.

Three main types of G:(1) Gs (activation of adenylate cyclase)(2) Gi/o (inhibition of adenylate cyclase,

activation of phosphodiesterase)(3) Gq (activation of phospholipase C)

GIRK Activation

Kandel et al., Principles of Neural Science,5th Edition, Figure 11-12

Free remains tethered to the membrane near the receptor and can only activate nearby channels (spatially restricted).

Effector Pathways Associated with GPCRs (Fig. 7.6)

Tools to study G protein signaling (applied intracellularly):

Cholera Toxin: Prevents GTP bound Gs subunit from GTP->GDP hydrolysis (prolonged Gs activation)

Pertussis Toxin: Locks Gi subunit in its GDP bound form (prevents Gi activation)

GTPS: Non-hydrolyzable GTP analog. Prevents activated G protein from being inactivated.

GDPS: Non-hydrolyzable GTP analog. Prevents activated G protein from being inactivated.

GTPS

GDPS

Types of GTP-binding Proteins: Small G Proteins (Fig. 7.5)

Monomeric G-proteins (small GTPase): acts like a monomeric subunit to regulate intracellular signaling events (Ras family of small GTPase regulates cell proliferation during development and also mediate long-term synaptic potentiation).

GTP-binding protein regulators: GEFs & GAPs.

GEFs: catalyzes the dissociation of GDP, allowing a GRP molecule to bind to the G protein. The localization of GEFs can determine where a certain small G protein will be active. GEFs are often recruited by adaptor proteins in response to upstream signals. For example, activation of EGF receptor recruit an adaptor protein, GRB2, which recruit SOS1 (a Ras GEF) to the membrane, where SOS1 can activate the membrane bound Ras.

GEFs: Guanine nucleotide exchange factors

GAPs: GTPase activating proteins (the endogenous GTPase activity of the G protein is usually too slow to terminate the signal)

Neuronal Second Messengers (Fig. 7.7B-D)

Ca2+-induced Ca2+ release (CICR)

Calcium: extracellular and/or intracellular calcium: most common & important 2nd messenger in neurons.

(Calcium-imaging has become an extremely popular method to measure neuronal activity.)

Cyclic nucleotides (cAMP or cGMP): Gs, Gi/o signaling

DAG & IP3 : Gq signaling

10-3 M

10-8 M~10-7 M

Membrane phospholipid

Neuronal Second Messengers (Fig. 7.7A)

Gs

Gi/o

Gq

Extracellular Ca2+

Intracellular Ca2+

Different PLC isoforms are differentially regulated by Gq, Ca2+, and receptor tyrosine kinase

Requires ATP

Powered by Na+ gradient

Regulation of Cellular Proteins by Phosphorylation (Fig. 7.8)

Protein kinases transfer phosphate groups from ATP to serine, threonine or tyrosineon substrate proteins (aa with a hydroxyl group). Ser/Thr kinases (usually activated by 2nd messengers) or Tyr kinases (usually activated by extracellular signals, e.g. NGF/TrkA). How do you know a protein has a potential to be phosphorylated?

Phosphorylation reversibly alters the structure and function of cellular proteins.

Removal of the phosphate groups is catalyzed by protein phosphatases (Ser/Thrphosphatases, calcineurin, etc), which can also be regulated by 2nd messengers or be constitutively active.

Substrate proteins:Enzymes, Neurotransmitter Receptors,Sensory Receptors, Ion Channels, and Structural Proteins.

Higher substratespecificity

Less substratespecificity (not as many types as kinases)

Phosphorylation Sites

(Mendez et al., Neuron 28, 153-164, 2000)

Mechanism of Protein Kinase Activation (Fig. 7.9)

PKA: a Ser/Thr kinase, two catalytic subunits & two inhibitory (regulatory) subunits. cAMPbinds to the inhibitory subunits, exposing the catalytic subunits (disinhibition, a common mechanism for kinase activation).

CaMKII: a Ser/Thr kinase, composed of many subunits (8-14). Each subunits contains a catalytic, a regulatory (auto-inhibitory) and a self-association domain. Ca2+/CaM binding releases the catalytic domain. CaMKII can autophosphorylate itself (blocks auto-inhibition, persistent activation, important for long-term potentiation to strengthen synaptic connection).

PKC: a monomeric Ser/Thr kinase, DAG causes PKC to translocate to the membrane, where PKC binds to Ca2+ and phosphatidylserine (PS) to disinhibit the kinase.

(cAMP-dependent protein kinase)

(calcium/calmodulin-dependent protein kinase II )

(protein kinase C)

Regulation of Tyrosine Hydroxylase by Phosphorylation (Fig. 7.14)

Tyrosine hydroxylase: controls the rate-limiting step of catecholamine neurotransmitter synthesis (dopamine, norepinephrine & epinephrine).

TH activity can be regulated by several kinases, including PKA, CaMKII, MAP kinases and PKC (2nd

messangers: cAMP, Ca2+, and DAG).

Receptor Tyrosine Kinases

Kandel et al., Principles of Neural Science, 5th Edition, Figure 11-9

Ligand bindingDimerizationPhosphorylation of its partner

Receptor auto-phosphorylationRecruitment of adaptor proteinsDifferent downstream signaling events

Mitogen-activated protein kinase (MAPK): activate other kinases by direct phosphorylation (MAPKK or MAP2K, MAP3K, MAP4K)

GRB2

GEF

Tyrosine Kinases (Fig. 7.12)

Two types: receptor (RTKs) and non-receptor Tyr kinases (e.g. Src kinase).

Tyr phosphorylation is less common than Ser/Thr phosphorylation and often serves to recruit signaling molecules to the phosphorylated protein.

Receptor tyrosine kinase: activated by extracellular molecules (e.g. NGF).

Transmembrane proteins: extracellular ligand-binding domain (growth factors, neurotrophic factors or cytokines) and intracellular catalytic domain.

NGF: nerve growth factor (neurotrophin)

TrkA: Tyrosine receptor kinase

NGF binding causes TrkA to dimerize and phosphorylate its partner (auto-phosphorylation). Phosphorylated TrkA serves to tether various adapter proteins or PLC to activate multiple parallel signaling events (PI3 kinase, ras signaling, and PLC pathway).

Nuclear Signaling/Transcription (Fig. 7.10)

Receptors for membrane-permeant agonists (e.g. steroids): intracellular. Upon ligand binding, the receptor translocate into nucleus to trigger transcription.

Thyroid hormone: receptors is bound to DNA to repress transcription in the absence of TH.

Some membrane receptors can also trigger intracellular signaling cascade to reach the nucleus.

Features: act on a longer time scale (30-60 minutes to transcribe/translate new RNA or proteins, even longer (hours to days) to reverse the events.

Can lead to systemic, permanent change of a neuron (neuronal differentiation).

Regulation: activation, translocation, transcription or translation of transcription factors.

Example: CREB (cAMP response element binding protein), immediate early genes (e.g. c-fos) and delayed response genes.

1. Decondensation of chromatin to provide binding sites for transcription factors

2. Transcription factor binding promote unwrapping of DNA to expose the promoter region upstream the coding region

3. RNA polymerase assembles on the promoter and begins transcription

Upstream activator site

Transcriptional Regulation by CREB (Fig. 7.11)

CREB: cAMP response element binding protein, a transcription factor, which is activated by phosphorylation.

CREB normally binds to CRE on DNA (as homodimer or heterodimer).

Phosphorylated CREB become an activated transcription factor, which then stimulate RNA polymerase to begin transcription.

Multiple pathways can lead to CREB phosphorylation: PKA, CaMKIV, ras/MAP signaling.

Phosphorylation of CREB must be maintained long enough to ensure transcription (because of basal phosphatase activity). Convergence of multiple signaling pathways.

Can result in long-lasting changes (learning & memory).

CREB target genes: immediate early genes (e.g. c-fos), neutrotrophin BDNF, tyrosine hydroxylase (to make DOPA for dopamine synthesis), and many neuropeptides.

Immediate early genes: transcription factors the expression of which is dramatically increased over 30-60 minutes after a cell is activated/stimulated.

Delayed response genes: genes which are turned on by immediate early genes.

Background: Target tissues of sympathetic and sensory neurons secret NGF to promote nerve growth/differentiation and survival. The neurotrophic effect of NGF requires activation of CREB transcription factor at the cell body. However, it is unclear how NGF signaling at the axon terminal is propagated back to the soma which can be over 1 meter away. Is NGF internalized by itself with activated TrkA back to the cell body for the transmission of the signal? Alternatively, is some downstream signaling proteins (ras, MAPK?) or 2nd

messengers generated at the axon terminal upon NGF-TrkA interaction transported back to the cell body?

Experiments: Use compartmentalized cultures of sympathetic neurons (axon terminals and cell bodies are cultured in different chambers). Generate phospho-specific antibodies that label site specific-phosphorylated and unphosphorylated CREB (anti-P-CREB) and TrkA (anti-P-TrkA). This system enables exposure of terminals or cell bodies to NGF and then to assess by immunocytochemistry the phosphorylation (activation) state of CREB and TrkAin cell bodies.

1. Maintain the culture in low NGF (2 ng/ml).2. Stimulate the culture with high NGF (200 ng/ml) at

either axon terminals or cell bodies.3. Immuno-stain with anti-P-CREB and/ot anti-P-TrkA4. Speed: 2-4 mm/hour

Whole-cell lysates (controls)

Cell bodies

Axon terminals and distal processes

Cell bodies

Axon terminals and distal processes (center-plated)

Side-plated

Fig. 1. Phosphorylation of CREB after application of NGF to sympathetic neurons

Campenot Chamber

anti-P-CREB

Fig. 2. Internalization of NGF is required for CREB activation at cell bodies

NGP-coupled microbeads: can activate TrkA but can not be internalized

anti-P-TrkA

Results: Internalization of NGF and its receptor TrkA, and their transport to the cell body, were required for transmission of the transmission of target-derived NGF signaling. The tyrosine kinase activity of TrkA was required to maintain it in an auto-phosphorylated state upon its arrival in the cell body and for propagation of the signal to CREB within neuronal nuclei. Thus the NGF-TrkAcomplex is a messenger that delivers the NGF signal from axon terminals to cell bodies of sympathetic neurons.