VI b Cell Signalling

Second Messenger System

• When a ligand binds to a G protein-coupled receptor (GPCR), the GPCR activates a G protein inside the cell.

• The G protein then activates an effector enzyme, which generates a second messenger.

Activation of G Protein-Coupled Receptors

• Activation of GPCRs by a ligand activates a G protein.

• The G protein activates enzymes called G protein-regulated enzymes.

• All G protein-regulated enzymes are associated with the production of a second messenger.

Second Messengers

• Intracellular messengers triggered by a first messenger (ligand) activating a receptor, which then activates a G protein.

G Proteins and Amplifier Enzymes

• Activated G proteins activate amplifier enzymes (effectors).

• Amplifier enzymes generate second messengers.

Activation Process

1. A messenger binds to a receptor.

2. This activates a G protein.

3. The G protein activates an amplifier enzyme.

4. The amplifier enzyme produces a second messenger.

Types of G Proteins

• Gs (G stimulatory protein): Activates amplifier enzymes, leading to the production of a second messenger.

• Gi (G inhibitory protein): Inhibits amplifier enzymes, inhibiting the production of a second messenger.

Role of Second Messengers

• Amplify the signal of the ligand.

• One signal binds to a receptor, activating a G protein, which then activates the production of many second messengers.

Types of Second Messengers

1. Cyclic AMP (cAMP)

   • Enzyme: Adenylate cyclase

   • Substrate: ATP

   • Action: Activates protein kinase A (PKA), a serine/threonine protein kinase, $(\text{phosphorylates proteins on serine/threonine residues})$. This changes their activity and brings about a response by the cell.

2. Cyclic GMP (cGMP)

   • Enzyme: Guanylyl cyclase

   • Substrate: GTP

   • Action: Activates protein kinase G (PKG), which phosphorylates target proteins on serine/threonine residues, activating these proteins and bringing about a response.

3. Calcium Ion ($Ca^{2+}$)

   • Increased calcium ion concentration inside the cell can cause a response (e.g., muscle contraction, increased exocytosis).

   • Calcium can bind to calmodulin, and the calcium-calmodulin complex can activate specific kinases, which then phosphorylate other proteins, changing their activity and bringing about a response.

4. Inositol Triphosphate (IP3) and 5. Diacylglycerol (DAG)

   • Generated from the hydrolysis of phosphatidylinositol bisphosphate (PIP2) by phospholipase C.

   • $(\text{Phospholipase C hydrolyzes PIP2 to generate IP3 and DAG})$.

   • IP3 binds to receptors on the endoplasmic reticulum (ER), causing calcium release inside the cell, leading to a response.

   • DAG activates protein kinase C (PKC), which phosphorylates proteins on serine/threonine residues, causing a response by the cell.

Summary Table of Second Messengers

The following table summarizes the types of second messengers, their precursors, the enzymes involved, their actions, and the first messengers that activate the GPCRs leading to their synthesis:

Examples of Second Messenger Systems

Cyclic AMP (cAMP) System

1. A messenger binds to a GPCR.

2. The GPCR activates an intracellular G protein.

3. The alpha subunit of the G protein activates adenylate cyclase.

4. Adenylate cyclase synthesizes cAMP from ATP.

5. cAMP activates protein kinase A (PKA), leading to a cellular effect.

6. Phosphodiesterase hydrolyses cAMP to AMP, terminating the signal.

Steps of cAMP Generation

1. A first messenger binds to a GPCR.

2. The activated receptor activates a G stimulatory protein (Gs).

3. The alpha subunit of Gs, bound to GTP, activates adenylate cyclase.

4. Adenylate cyclase converts ATP into cAMP.

5. cAMP activates protein kinase A.

6. PKA phosphorylates many proteins on serine and threonine residues by transferring a phosphate group from ATP.

7. Phosphorylation alters protein activity, causing a response by the cell.

Regulation of Adenylate Cyclase

• Binding of an extracellular signal or messenger to the receptor activates the trimeric G protein.

• This leads to the dissociation of the alpha subunit, which activates adenylate cyclase.

• Adenylate cyclase generates cAMP from ATP.

• cAMP activates PKA, which phosphorylates many target proteins on their serine and threonine residues.

• This net change in phosphorylation in the cell brings about a response, amplifying the signal.

Step-by-Step Diagram of cAMP Second Messenger System

1. GPCR is linked to a trimeric G protein.

2. Before the messenger binds, the G protein is inactive, bound to GDP.

3. Binding of the messenger activates the G protein.

4. GDP is exchanged for GTP, leading to dissociation of the alpha subunit.

5. The alpha subunit activates adenylate cyclase.

6. Adenylate cyclase generates cAMP from ATP.

7. cAMP activates PKA.

8. PKA phosphorylates many proteins on serine and threonine residues, bringing about a response in the cell.

Cellular Processes Regulated by cAMP

• Transport

• Microtubule assembly/disassembly

• Protein synthesis

• Gene expression

• Glycogen synthesis/breakdown

• Triglyceride breakdown and formation of fatty acids

Cholera Toxin and G Proteins

• Cholera toxins, released by Vibrio cholerae bacteria, bind to a membrane ganglioside on secretory cells in the small intestine.

• The A subunit of the toxin enters the cell and activates a G protein, causing sustained activation.

• The alpha subunit dissociates and activates adenylate cyclase, which catalyzes the formation of cAMP from ATP.

• cAMP activates PKA.

• PKA phosphorylates target proteins, enhancing the secretion of chloride ions out of the cell.

• The flow of chloride ions out of the cell causes sodium ions to follow, and water follows by osmosis, resulting in severe diarrhea.

Phosphatidylinositol Second Messenger System

• The membrane phospholipid PIP2 is hydrolysed by phospholipase C, generating diacylglycerol (DAG) and inositol triphosphate (IP3).

Steps of the System

1. A ligand binds to a GPCR.

2. The receptor activates a G protein.

3. The alpha subunit of the G protein activates phospholipase C.

4. Phospholipase C hydrolyzes PIP2, generating DAG and IP3.

5. IP3 binds to receptors on the endoplasmic reticulum (ER), causing the release of calcium ($Ca^{2+}$) from the ER, which can be considered as a third messenger.

6. Calcium can bind to calmodulin, and this complex activates protein kinases, leading to phosphorylation and a change in the response.

7. DAG directly activates protein kinase C (PKC), which phosphorylates other proteins on serine/threonine residues.

Diagram of Phosphatidylinositol Second Messenger System

• When the ligand binds to the receptor, it activates the G protein, leading to the displacement of GDP, replacing it by GTP.

• The alpha subunit dissociates and activates phospholipase C.

• Phospholipase C hydrolyzes PIP2 into IP3 and DAG.

Detailed Diagram Explanation

1. The G protein coupled receptor is present.

2. Before a messenger binds, the inactive G protein is bound to GDP.

3. When the messenger binds, it activates the G protein.

4. Activation leads to replacement of GDP by GTP.

5. The alpha subunit dissociates and activates phospholipase C.

6. Phospholipase C hydrolyzes PIP2, generating DAG and IP3.

7. DAG activates protein kinase C, which phosphorylates target proteins on serine/threonine residues, leading to a cellular response.

8. IP3 binds to receptors on the ER, causing the release of calcium from the lumen of the ER into the cell.

9. Increased calcium can lead to a response (e.g., muscle contraction, exocytosis).

10. Calcium can also act as a messenger by binding to calmodulin.

11. The calcium-calmodulin complex activates other protein kinases, leading to changes in contraction, metabolism, or transport.

Significance of Second Messenger Systems

• Signal Amplification: A small amount of ligand can cause a huge response in the target cell.

• Each step recruits more participants.

  • Ligand binds receptor → receptor recruits G protein → G protein activates adenylate cyclase → adenylate cyclase generates cAMP → cAMP activates kinases, etc.

Characteristics of Second Messenger Systems

• One messenger binds to one receptor, activating multiple G proteins.

• The alpha subunits activate adenylate cyclase, generating hundreds of cAMP molecules.

• Each cAMP activates protein kinase A, and each PKA phosphorylates hundreds of proteins.

• One messenger molecule leads to the phosphorylation of millions of proteins, amplifying the signal.

Summary of Cell Signaling Lectures

• Types of communication: direct and indirect.

• Principles of cell signaling.

• Players in cell signaling: messengers, receptors.

• Types of receptors: intracellular and cell surface.

• G protein-coupled receptors (GPCRs).

• Second messenger systems: cAMP and phosphatidylinositol systems.

• Activation of GPCRs leads to the activation of effector enzymes, which generate second messengers.