Chapter 15: Signal transduction

Know the terminology:Enzyme-linked receptor, G-protein linked receptor, nuclear hormone receptor, G-protein, adaptor protein, scaffolding protein, SH2 domain, MAPK, Ras, protein kinase, MAPK, protein phosphatase, phospholipase, phosphodiesterase, cAMP, crosstalk,

Chapter 15: Signal transduction Outline:General principles of signal transductionOverview of:

SignalingReceptorsTransducersTargets

Major types of cell-surface receptorsRTK signalingG-protein signaling

General Principles of Signal Transduction

1. Communication usually involves a (i) signaling molecule, (ii) a receptor, (iii) intracellular signal transducers and (iv) targets

General Principles of Signal Transduction2. Each cell responds to a complex profile of signaling molecules (crosstalk)

General Principles of Signal Transduction3. Different cells respond differently to a

particular signaling molecule

General Principles of Signal Transduction4. Cells can remember the effects of some signals

5. Cells can adjust their sensitivity to a signal

General Principles of Signal Transduction4. Cells can remember the effects of some signals

5. Cells can adjust their sensitivity to a signal

General Principles of Signal Transduction6. Signal can exhibit complex responses to signal

concentration

Signaling molecules

Signaling molecules come in many chemical forms:• Proteins: insulin, glucagon• Steroids et al.: testosterone, estradiol, cortisol• Amines: thyroxine, catecholamines, acetylcholine • Gases: nitric oxide

Signaling pathways require molecules with rapid rates of synthesis and degradation

Typically released from one cell and recognized by another cell

Signaling molecules

Secretory signals:• Autocrine-signals affect same cell or cell type• Paracrine-signals affect neighbouring cell• Endocrine-signals affect distant cells

Contact-dependent signals:-signals are not released but affect other cells in

contact through protein-protein interactions

Autocrine signaling

Signals released by one cell affect other cells in the immediate vicinity

Amplify a response by inducing many “like-cells” to respond in the same way

Allows cells to exhibit a coordinated response (a community effect)

Autocrine signaling

Paracrine signaling

Signals released by one cell affect different cells in the immediate vicinity

Synaptic transmission resembles paracrine stimulation but the response is limited to cells in very close proximity

The outward propagation of the signal is limited by cellular uptake, extracellular degradation, and binding

Endocrine signaling

Signals released by one cell affect different cells far away

Endocrine signaling often exerts multiple effects on the organism by affecting many different tissues

Receptors

Proteins that bind signals and initiate a signaling cascade

Cell membrane receptors-integral membrane proteins that bind an extracellular signal and start a signal cascade

Intracellular receptors-nuclear hormone receptors

Nuclear hormone receptors

Examples that we have already discussed include steroid hormone receptor and thyroid hormone receptor

NHRs are transcription factors that respond to specific ligands

Ligands alter the ability to bind to specific DNA regulatory elements

Intracellular signal transduction

Once the receptor is activated, the signal is propagated by proteins that act as:

–Relay proteins–Messenger proteins–Adaptor proteins–Amplifier proteins–Transducer proteins–Bifurcation proteins–Integrator proteins–Latent gene regulatory proteins

Intracellular signal transduction

Activated cell membrane receptors can alter the activity of intracellular enzymes including:

–Protein modifying enzymes•kinases/ phosphatases•acetylases/ deacetylases

–Lipid modifying enzymes•Phospholipases•Phosphotidyl inositol kinase

–Nucleotide modifying enzymes•cyclases/ phosphodiesterases

Protein kinases

Phospholipases

PLC generates DAG and phosphoinositides, such as IP3 (inositol 1, 4, 5-triphosphate)

Targets

The final targets of signaling cascades are usually proteins: •Regulators of gene expression (transcription factors, histone remodeling enzymes)•Enzymes (metabolic enzymes)•Structural proteins (cytoskeletal proteins)

•Effects alter activity (catalytic, DNA binding) or the ability to interact with other proteins (structural proteins, subcellular localization).

Cell surface receptors3 main classes of cell surface receptors:

Ion-channel linked receptorsEnzyme linked receptors may possess intrinsic enzyme activity or, once ligands bind, activate enzyme activity

G-protein linked receptors are trimeric GTP-binding protein (G-protein) that regulate the activity of other proteins

Enzyme-linked receptors5 main classes distinguished by:•type of effector (e.g. kinase vs. phosphatase) •target (serine/threonine, tyrosine, histidine)•type of linkage between receptor and enzyme

1. Receptor tyrosine kinase (-RTK)2. Tyrosine kinase linked receptor3. Receptor serine/threonine kinase4. Receptor guanylyl cyclase5. Histidine-kinase associated receptors

Receptor tyrosine kinasesMost common type of receptor for many common protein hormones including EGF, PDGF, FGF, HGF, IGF-1, VEGF, NGF.

Receptor tyrosine kinasesReceptor itself possesses intrinsic tyrosine kinase activity

Once the ligand binds, the receptor can dimerize and it become an active tyrosine kinase

It phosphorylates itself (autophosphorylation), causing:

1. Increase kinase activity2. Increased affinity for other proteins

Once bound, these docking proteins can become phosphorylated

Ligand-dependent autophosphorylation and docking

Ligand-dependent autophosphorylation and docking

Docking of intracellular proteins on phosphotyrosines

Phosphotyrosine domains are binding sites for many different proteins with SH2 (=PTB) domains

These can be enzymes (e.g., PLC, PI3K) or they can act as adaptor molecules to bind other proteins

Linking RTK to Ras and the MAPK cascadeOnce an adaptor protein (e.g., Grb2) binds to the RTK, it attracts another protein - Ras GEF (guanine nucleotide exchange factor)

Ras GEF induces Ras to exchange its GDP for GTP (activating Ras).

Active Ras then activates MAPKKK, which phosphorylates and activates MAPKK, which phosphorylates and activates MAPK, which phosphorylates many proteins, including transcription factors.

Activation of Ras

Activation of MAPK cascade

Scaffolding proteins help organize MAPKs

Summary: Enzyme-linked receptors

How do enzyme-linked receptors generate variable cellular responses?

Multiplicity of players (receptors, kinases etc) arise from gene duplication and divergence

Recognize the critical role of phosphorylation/ dephosphorylation control as molecular switches

Adaptor molecules allow construction of protein signaling cascades with variable outputs

G-protein linked receptorsLigand: Diverse ligands, such as epinephrine

Receptor: Integral membrane protein with 7-transmembrane domains

G-protein: Trimeric protein (α, β, γ) attached to the cell membrane by lipid anchors

Effectors: Target proteins that show altered activity when they interact with activated G-protein subunits (α, or βγ)

G-protein linked receptors and G-proteins

Receptor

G-protein

Interaction between receptor and G-protein

Once the ligand binds, the activated receptor recruits a G-protein

Nucleotide exchange occurs (GTP replaces GDP) and the trimer dissociates into 2 parts:

-α subunit-βγ subunit

Both parts can regulate downstream pathways

G-protein dissociation

GTP hydrolysis ends signaling and induces trimerization

Gs proteins are stimulatory

Upon dissociation, a Gs protein stimulates an effector enzyme, such as adenylate cyclase

Adenylate cyclase converts ATP to cAMP

Elevated cAMP stimulates cAMP-dependent protein kinase (PKA) by inducing the release of inhibitory subunits

PKA activation by cAMP

PKA activates gene expression

Inactivation of PKA pathway

The G-protein -PKA pathway is inactivated by:•Receptor desensitization•GTP hydrolysis in G-protein•cAMP hydrolysis by phosphodiesterase•PKA inhibition•Phosphatase action on PKA targets•Activation of an antagonistic pathway (Gi)

G-proteins and phospholipases

Some G-proteins activate PLC (phospholipase C), triggering formation of inositol triphosphate (IP3) and diacylglycerol (DAG)

DAG, IP3, Ca2+ and signal transductionDAG:•substrate for production of eicosanoids, potent signaling molecules including arachadonic acid•activates PKC

IP3:induces release of Ca2+ from ER stores via IP3-sensitive Ca-channels

Ca2+:Elevated Ca2+ can activates PKC and CamK.

Interactions between G-proteins and RTKs