Signal transduction

In signal transduction, extracellular signals are converted into intracellular signals: A signaling molecule (ligand) reaches its target cell and binds to a specific receptor. This activates a signaling cascade involving intracellular enzymes and molecules (second messengers), which again leads to a specific reaction. Via signal amplification, the number of signaling molecules is increased at every step of the signal cascade.

• Receptor: Proteins that receive signals from outside of the cell by binding specific ligands, thereby initiating an intracellular signaling cascade.
  • Cell surface receptors (membrane receptors): located in the cell membrane (e.g., G protein-coupled receptors)
  • Intracellular receptors: located inside the cell (e.g. steroid receptors)
    • Nuclear receptors (ligand-dependent transcription factors): intracellular receptors that act inside the nucleus
• Ligand (first messenger): A chemical messenger that binds specifically to one receptor (e.g. proteins, steroids, hormones, neurotransmitters, small organic molecules such as nitric oxide (NO)).
• Second messenger: small molecules (e.g. cGMP, IP₃, Ca²⁺ ions) that mediate the intracellular response to an extracellular stimulus
• Signaling cascade: The interconnection of individual steps in a signaling pathway.
• Signal amplification: The increase in the number of signaling molecules at every step of the signal cascade.

Overview

• Extracellular messengers have to bind to a receptor to exert their effect.
• Lipophilic messengers can pass through the cell membrane and bind to intracellular receptors, while hydrophilic messengers cannot due to the lipophilic properties of the cell membrane.
• Therefore, hydrophilic messengers typically act on integral membrane receptors, which translate the signal of the extracellular messenger into an intracellular signal.

Intracellular receptors

• Definition: receptors that are located inside the cell
• Examples of effectors
  • IP3-receptor: in the endoplasmic reticulum
  • Nuclear receptors: ligand-dependent transcription factors that act within the nucleus
    • Activation principle
      1. A lipophilic hormone diffuses through the cell membrane.
      2. Hormone and receptor form a complex.
      3. Hormone-receptor complex binds to hormone-responsive elements (specific DNA sequences that are able to bind complementary hormone-receptor complexes) in the nucleus and acts as a transcription factor.
    • Location prior to ligand binding
      • Cytosol (e.g., mineralocorticoid, glucocorticoid, estrogen, progesterone, testosterone receptors)
      • Nucleus (e.g., thyroid hormone, vitamin D receptors)
• Examples of ligands
  • Cortisol
  • Aldosterone
  • Testosterone
  • Estrogen
  • Progesterone
  • Thyroxine, triiodothyronine
  • Vitamin D

Ligands of the intracellular receptors (Progesterone, Estrogen, Testosterone, vitamin D, Cortisol, Aldosterone, Thyroid hormones): PET Da CAT

Cell surface receptors

• Hydrophilic hormones transmit signals by binding to receptors present in the cell membrane (= cell surface receptors).
• There are three types of cell surface receptors:
  • G protein-coupled receptors
  • Enzyme-linked receptors
  • Ligand-gated ion channels

G protein-coupled receptors

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors. The action of GPCRs depends on three elements: the receptor, the G protein, and the effector molecule.

• Examples of ligands: catecholamines, anterior pituitary hormones (ACTH, LH, FSH, TSH), glucagon
• Receptor structure
  • Receptor with seven transmembrane helices
  • Binding sites for ligands are found in extracellular regions or between helices.
  • Has an intracellular binding site for the G protein
• G protein
  • A heterotrimeric protein composed of three subunits
    • α subunit
      • Binds GDP in the inactive state and GTP in the active state
      • GTPase activity: hydrolyzes GTP to GDP and phosphate, thereby terminating α subunit activity
    • β subunit: stable complex with the γ subunit
    • γ subunit: complex with the β subunit with a lipid anchor in the cell membrane
  • Activation principle
    1. The binding of an extracellular ligand causes a conformational change in the receptor.
    2. The receptor binds intracellularly to G protein.
    3. Activated G protein binds GTP instead of GDP.
    4. Three subunits of the G protein dissociate in a complex composed of β and γ subunits and the α subunit.
  • GTPase: a small G protein composed of only an α subunit that functions independently to hydrolyze GTP to GDP and phosphate
• Effector molecules
  • Activation: by the α subunit of the G protein
  • Function: synthesis of second messengers (see below) depending on which G protein is coupled to the receptor
    • G_s proteins: stimulate adenylyl cyclase
    • G_i proteins: inhibit adenylyl cyclase
    • G_q proteins: stimulate phospholipase c

G_s proteins activate adenylyl cyclase, whereas G_i proteins inhibit adenylyl cyclase.

Receptor tyrosine kinases (RTKs)

Receptor tyrosine kinases are transmembrane receptors that are generally activated by ligand-induced dimerization and autophosphorylation of cytoplasmic tyrosine residues, which triggers activation of downstream signaling cascades.

• Examples of ligands: insulin, growth factors (e.g., EGF, IGF, FGF, TGF)
• Receptor structure:
  1. Extracellular domain
  2. Single transmembrane domain
  3. Intracellular domain with tyrosine kinase activity
• Activation principle
  1. Ligand binding to the extracellular domain results in receptor dimerization.
  2. The two adjacent tyrosine kinase domains phosphorylate one another to tyrosine residues (autophosphorylation).
  3. Increased kinase activity through autophosphorylation
  4. A number of different signal transduction molecules with SH₂ domains bind to the phosphorylated tyrosine residues and are activated → activation of various effectors of different signaling pathways
• Examples of effectors
  • Phospholipase C
  • Ras
    • Monomeric, membrane-bound GTPase
    • Activated Ras activates further signal transduction pathways such as the mitogen-activated protein kinase (MAPK) cascade → transcription of target genes for cell growth and proliferation
    • The Ras genes (KRas, HRas, NRas) are protooncogenes
    • Examples of signaling pathways with Ras involvement: insulin, platelet-derived growth factor (PDGF)

Ligands of the tyrosine kinase receptors (TYRosine kinase, INSULin, GROWth factors): “I'm TYRed of your INSULts, GROW up!”

Non-receptor tyrosine kinases

• Examples of ligands: growth hormone, prolactin, erythropoietin, thrombopoietin, cytokines (e.g., G-CSF, IFN, IL-2, IL-6)
• Receptor structure
  • Membrane receptor without tyrosine kinase activity
  • Tyrosine kinase from the Janus kinase (JAK) family is coupled as a separate protein to the receptor.
• Activation principle (similar to receptor tyrosine kinases)
  1. Ligand binding leads to receptor dimerization.
  2. Two neighboring tyrosine kinase domains of JAK phosphorylate each other (autophosphorylation) → JAK activation
  3. Development (at the phosphorylated tyrosine residues) of binding sites for SH₂ domains of signal proteins (STAT proteins).
  4. STAT proteins are phosphorylated and dimerized by JAK.
  5. STAT dimers exert their effect directly in the nucleus as a transcription factor for JAK-STAT regulated genes.

Ligands of the non-receptor tyrosine kinase (Prolactin, Interleukins/IFN, Growth hormone, G-CSF, Erythropoietin, Thrombopoietin): PIGGLET

Receptor serine/threonine kinases

• Examples of ligands: TGFβ (transforming growth factor β)
• Receptor structure: two subunits (type I and type II receptors) each with serine/threonine kinase activity
• Activation principle
  1. Ligand binding
  2. Receptor oligomerization
  3. The type II receptor activates the type I receptor.
  4. The type I receptor phosphorylates SMAD proteins.
  5. SMAD serves as a transcription factor for target genes.

Ligand-gated ion channels

Since most ligands of ligand-gated ion channels are neurotransmitters, only a short overview is provided here.

• Examples of ligands: acetylcholine, GABA, glutamate, IP₃
• Receptor structure: Cell surface receptors that act as ion channels
• Activation principle (example)
  1. Ligand binding → conformational change
  2. Pore opening enables ion movement (Na⁺, K⁺ Ca²⁺, Cl^-) across the cell membrane.
  3. Altered membrane potential → rapid signal response

Second messengers are small molecules that mediate the intracellular response to an extracellular stimulus.

cAMP (cyclic adenosine monophosphate) and protein kinase A

Synthesis

• A membrane-bound adenylyl cyclase synthesizes cAMP from ATP
• Signal amplification: An activated adenylyl cyclase enzyme can synthesize thousands of cAMP molecules.
• Regulation of adenylyl cyclase: depends on the type of G protein of the G protein-coupled receptor
  • G_s proteins: activate adenylyl cyclase → ↑ cAMP
  • G_i proteins: inhibit adenylyl cyclase → ↓ cAMP

Effects

cAMP activates ; protein kinase A (PKA).

• Enzyme class: serine/threonine kinase
• Activation: cAMP leads to the release of catalytic subunits of PKA
• Mechanism: : PKA controls the activity (activation or inactivation) of numerous enzymes via phosphorylation of serine and threonine residues.
• Example: glycogen metabolism to ↑ blood glucose concentration
  • Hypoglycemia → ↑ glucagon or adrenaline → GPCR activation → ↑ cAMP → activation of PKA, which causes:
    • Phosphorylation of glycogen phosphorylase → activation → increased mobilization of glucose from glycogen
    • Phosphorylation of glycogen synthase → inhibition→ decreased glycogen formation

Degradation

cAMP is degraded by phosphodiesterase to adenosine monophosphate (AMP).

Examples of ligand hormones

• Hypothalamic hormones: GHRH, CRH, vasopressin (V2 receptor)
• Pituitary hormones: TSH, ACTH, FSH, LH, MSH
• Other hormones: PTH, calcitonin, glucagon, histamine (H2 receptor), hCG

cGMP (cyclic guanosine monophosphate)

• Synthesis: cGMP is synthesized from GTP by the guanylate cyclase. There are two subforms of guanylate cyclase:
  1. Soluble guanylate cyclase: activated by nitric oxide
  2. Membrane-bound guanylate cyclase: activated through extracellular ligand binding (e.g., EDRF, ANP, BNP; )
• Effects
  • Activates cGMP-dependent protein kinase G in smooth muscle cells → inhibits Ca²⁺ outflow from the sarcoplasmic reticulum → ↓ intracellular Ca²⁺ → relaxed smooth vascular muscles → vasodilation
  • cGMP-dependent ion channels in the photoreceptor cells of the retina → preserve the unstimulated state → dark signal
• Degradation: by phosphodiesterase to GMP

Soluble guanylate cyclase is activated by nitric oxide.

The second messengers cAMP and cGMP are degraded and inactivated to AMP and GMP by various phosphodiesterases (PDE). A decrease in cAMP or cGMP causes contractions in smooth muscles. PDE inhibitors are used in the treatment of pulmonary hypertension (PDE-4 inhibitor roflumilast) and erectile dysfunction (PDE-5 inhibitor sildenafil).

Ligands of the guanylate cyclase (GMP, BNP, EDRF, ANP): GruMPy BEA

Nitric oxide

• Structure
  • Volatile gas: half-life of 2–30 s in the blood
  • Can freely diffuse across cell membranes, so NO can act as an intracellular and extracellular signaling molecule
• Synthesis: produced from L-arginine in two NADPH-dependent reactions, catalyzed by endothelial nitric oxide synthase (eNOS) in the endothelial cells of blood vessels
  • eNOS is stimulated by:
    • Physical effects such as arterial wall shear stress
    • Increase in intracellular calcium concentration in endothelial cells
    • Neurotransmitters and neuropeptides: e.g., acetylcholine, bradykinin, histamine, vasoactive intestinal peptide, and substance P
• Function: causes smooth muscle relaxation and subsequent dilation of blood vessels

Nitric oxide (NO) has a half-life of only a few seconds. It is not stored by the body but is synthesized as a result of activation. Nitrate drugs stimulate the formation and release of NO. Relaxation of smooth muscle cells in vessel walls leads to the dilation of coronary arteries, pulmonary arteries, and peripheral veins. Peripheral vasodilation leads to a decrease in cardiac preload.

IP₃ and DAG

• Synthesis
  1. Activation of a Gq protein-coupled receptor
  2. Activation of phospholipase C (an enzyme that cleaves phospholipids)
  3. Cleaving of the membrane-bound phospholipids PIP₂ (phosphatidylinositol 4,5-bisphosphate) into the second messenger IP₃ (inositol triphosphate) and DAG (diacylglycerol)
• Function
  • IP₃ (hydrophilic) diffuses into the cytoplasm → activation of IP₃ receptors at the membrane of the endoplasmic reticulum (ER) → Ca²⁺ release from the ER via the IP₃ receptor-coupled calcium channel → ↑ intracellular Ca²⁺ concentration → smooth muscle contraction
  • DAG (lipophilic) remains in the membrane → activates protein kinase C (PKC)
    • PKC is Ca²⁺-dependent (depends on IP₃-mediated Ca²⁺ release)
    • Mechanism of action: regulates the activity of various enzymes via phosphorylation of serine and threonine residues, e.g., regulatory proteins that influence cell growth (actin cytoskeleton) or differentiation (EGFR)
    • Examples of the effect of PKC: cell growth and proliferation
• Examples of ligands: vasopressin (V1 receptor), oxytocin, GnRH, TRH, angiotensin, gastrin, histamine (H1 receptor)

G_q proteins activate phospholipase C, which cleaves the second messengers IP₃ and DAG from PIP₂. These, in turn, activate PKC.

Ligands of the phosphoinositol signaling pathway (TRH, Vasopressin, Angiotensin, GnRH, Oxytocin, Gastrin, Histamine): TRy VAn GOGH

Ca²⁺ as a second messenger

• Principle
  • Intracellular Ca²⁺ levels are usually very low.
  • Second messengers (such as IP₃) or depolarization by action potentials result in opening of the Ca²⁺ channels in the membrane of cells or ER → Ca²⁺ spikes (= strong peak concentration)
  • Effect of Ca²⁺, especially as a complex with calmodulin: One molecule of calmodulin binds up to four Ca²⁺ ions → conformational change → regulation of calmodulin-dependent kinases/phosphatases, e.g., CaM kinase III (protein synthesis)
• Examples of Ca²⁺-mediated effects
  • ↓ Glycogen synthesis
  • ↓ Cholesterol synthesis

Ca²⁺ mediates the effect of other second messengers such as IP₃ and DAG via PKC activation. It also functions as a second messenger by acting directly, i.e., without activating another signaling molecule.