Case 4 - BBS2042

Case 4 - Membrane Receptors

1. Learning Goals

• Receptors: Understanding their role in signaling, including first and second messengers.

• Protein Communication and Recognition: Detailed mechanisms of signaling cascades through:

  • Post-Translational Modifications (PTMs)

  • Phosphorylation

  • Ubiquitination

• GCPR Types: Focus on the key functions and cascades of:

  • Gi

  • Gs

  • Gq

• Second Messengers: Importance of molecules like cyclic AMP, Diacylglycerol, Inositol trisphosphate, and Protein Kinase A (PKA).

• Feedback Mechanisms: Exploration of positive and negative feedback pathways with GPCRs.

• Tyrosine Kinase Receptors (TKRs): Structure, function, and their role in signaling.

• Calcium Signaling: Acting as a second messenger.

• Ligand Bias: How different ligands can influence outcomes in receptor signaling.

• Drugs: Investigate the roles of various drugs related to GPCR and TKR mechanisms.

2. Receptors – What Are They?

• Definition: Specialized proteins detecting extracellular signals (ligands) and converting them into intracellular responses.

• Ligands Can Be:

  • Hormones

  • Neurotransmitters

  • Growth factors

  • Cytokines

  • Sensory stimuli

• Main Functions:

  • Allow cells to sense their environment.

  • Facilitate communication between cells.

  • Enable adjustment in behaviors such as metabolism, gene expression, proliferation, and movement.

  • Essential for homeostasis and coordinated tissue function.

2.1 First Messengers

• Definition: Extracellular signaling molecules that bind to receptors.

• Characteristics:

  • Generally cannot cross the plasma membrane if hydrophilic.

  • Binds specifically to its receptor.

• Function:

  • Initiates the signaling cascade.

  • Does not enter the cell in GPCRs and RTKs.

  • Causes a conformational change in the receptor.

2.2 Second Messengers

• Definition: Small intracellular molecules transmitting signals from activated receptors to downstream targets.

• Importance:

  • Amplify and distribute the signal inside the cell.

  • Activate enzymes, ion channels, or transcription factors.

• General Properties:

  • Small and diffusible.

  • Rapidly produced and degraded.

  • Allow strong signal amplification.

3. General Principle: How Proteins Communicate

• Communication Mechanisms:

  1. Changing each other's structure.

  2. Modifying each other chemically.

  3. Regulated binding.

• Signaling Cascade: A stepwise process where:

  • Protein A activates protein B.

  • Protein B activates protein C.

  • The signal becomes amplified and regulated.

3.1 Conformational Changes (Allosteric Regulation)

• Process:

  • A ligand or protein binds.

  • Target protein changes shape (conformation).

  • Activity changes (on/off, increase/decrease).

• Key Concept: Structure determines function; small structural shifts can have significant functional consequences.

3.2 Post-Translational Modifications (PTMs)

• Definition: Chemical modifications added after protein synthesis acting like molecular switches or tags.

• Types:

  • Phosphorylation: Addition of a phosphate group by a kinase.

  • Importance:

    • Adds negative charge.

    • Alters protein shape and activity.

    • Creates docking sites for other proteins.

  • Functional effects include turning enzymes on/off and enabling signaling cascade propagation.

  • Reversibility: Kinases add, phosphatases remove phosphates, allowing tight regulation.

  • Ubiquitination: Attaching ubiquitin to a target protein.

  • Functions:

    • Marks for degradation.

    • Alters localization and activity.

    • Modifies interactions.

  • Signaling mechanism and regulatory processes.

  • Acetylation: Addition of an acetyl group, neutralizing lysine's positive charge, affecting interactions with DNA and proteins.

  • Methylation: Addition of methyl groups to lysine or arginine without charge change, altering shape and hydrophobicity.

  • Glycosylation: Covalent addition of carbohydrate chains to proteins, increasing size and stability.

  • Lipidation: Covalent attachment of lipid groups; increases hydrophobicity and anchors proteins to membranes.

3.3 Protein–Protein Interaction Domains

• Function: Act as molecular docking sites to ensure specificity and rapid protein interactions.

• Key Domains:

  • SH2 Domains: Recognize phosphorylated tyrosine residues.

  1. Phosphorylated by receptor tyrosine kinases.

  2. Recruits proteins to activated receptors.

  • PTB Domains: Bind short peptide motifs with flexibility in specificity.

  • SH3 Domains: Binds proline-rich sequences; independent of phosphorylation.

  • PH Domains: Recognize specific phosphoinositides in membranes, enhancing protein localization.

4. G-Protein–Coupled Receptors (GPCRs)

• General Structure: Integrate membrane proteins with seven transmembrane α-helices, connecting intracellular and extracellular domains.

• N-terminus: Located extracellularly; C-terminus is intracellular; vital for interaction with G-proteins.

• Function: GPCRs act as guanine nucleotide exchange factors for G-proteins.

4.1 Structure of Heterotrimeric G-Proteins

• Composition: Composed of three subunits - Gα, Gβ, Gγ.

• Inactive State:

  • Gα bound to guanosine diphosphate (GDP).

  • Gα associated with the Gβγ complex.

4.2 Step-by-Step Activation Mechanism

• Step 1: Ligand binds to GPCR causing conformational change.

• Step 2: GDP–GTP exchange occurs; GTP binds to Gα.

• Step 3: Activation allows Gα–GTP and Gβγ complex to interact with effector proteins, transmitting signal.

4.3 The Gαs Pathway

• Activation of Adenylyl Cyclase: Gαs–GTP activates adenylyl cyclase to convert ATP to cyclic AMP (cAMP).

• Properties of cAMP:

  • Small, diffusible, amplifies signal.

  • Activates downstream kinases including PKA.

4.4 The Gαi Pathway

• Effect: Inhibits adenylyl cyclase, reducing cAMP levels and subsequently lowering PKA phosphorylation of targets.

4.5 The Gαq Pathway

• Activation of Phospholipase C-β: Gαq–GTP activates PLC-β, cleaving PIP₂ to IP₃ and DAG.

• Calcium Release: IP₃ triggers calcium ion release from the endoplasmic reticulum, promoting downstream signaling through various kinases and pathways.

4.6 Termination of GPCR Signaling

• Mechanisms:

  • GTP hydrolysis returning Gα to GDP-bound state.

  • Degradation of second messengers such as cAMP.

  • Receptor desensitization through phosphorylation and internalization via β-arrestin.

5. Feedback Regulation of GPCR Second Messengers

• Importance: Second messengers regulate their own production usually through pathways like:

  • PKA activation of phosphodiesterases, reducing cAMP levels.

  • Direct phosphorylation of receptors and downstream proteins to reduce signaling.

6. Receptor Tyrosine Kinases (RTKs)

• Structure:

  • Extracellular domain (ligand binding), single transmembrane helix, intracellular tyrosine kinase domain.

6.1 Inactive State of RTKs

• Condition: Monomeric structure, inactive kinase domain with unphosphorylated tyrosines.

6.2 Step-by-Step Activation Mechanism

• Step 1: Ligand binding leads to receptor dimerization.

• Step 2: Transfer autophosphorylation (cross-phosphorylation) activating the kinase domain.

• Step 3: Phosphorylation of the activation loop stabilizes kinases, enhancing catalytic activity.

6.3 Creation of Docking Sites

• Function of Phosphorylated Tyrosine Residues: Serve as recognition sites for proteins with SH2 and PTB domains, facilitating specificity and signal integration.

6.4 Major Downstream Signaling Pathways

• Ras–MAPK Pathway: Key for gene expression and cell proliferation involving several phosphorylation cascades.

• PI3K–Akt Pathway: Regulates survival and metabolism by inhibiting apoptosis through activation of survival signals.

6.5 Termination of RTK Signaling

• Mechanisms: Include removal of phosphates by protein tyrosine phosphatases, receptor endocytosis, and ubiquitination that marks for degradation.

7. Calcium as a Second Messenger

• Ideal Characteristics:

  • Sustained low cytosolic concentration, allowing rapid influx upon channel opening.

  • Effective in signaling due to rapid onset and amplification of responses.

7.1 Calcium Signal Types

• Calcium signal encoding: Can involve amplitude, frequency, localization providing specificity in responses.

7.2 GPCR Calcium Signaling

• Gαq Pathway Activation:

  1. Ligand binding activates PLC-β, cleaving to produce IP₃ and DAG.

  2. IP₃ mediates calcium release, creating regenerative amplification.

8. Ligand Bias

• Definition: The phenomenon of different ligands preferentially activating specific downstream pathways when binding to the same receptor.

• Significance: Reveals that ligands do not uniformly activate receptors, allowing diverse signaling outputs.

9. Transactivation

• Mechanism: GPCR activation can lead to indirect RTK activation through the release of growth factor precursors via protease activation.

10. Drugs

10.1 GPCR-Related Drugs

• Selective β₂-Adrenoreceptor Antagonists: Used rarely, primarily experimental. Decreases Gs activity, causing bronchoconstriction.

• Antihistamines: Block H₁ receptors leading to reduced allergy symptoms; divided into sedating and non-sedating kinds.

• Phosphodiesterase Inhibitors: Amplify GPCR signaling by preventing breakdown of cAMP/cGMP, used in various therapeutic contexts like asthma and erectile dysfunction.

• GLP-1 Receptor Agonists: Enhance insulin secretion in response to glucose, used in Type 2 diabetes treatment.

10.2 TKR-Related Drugs

• Tyrosine Kinase Inhibitors: Common in cancer treatment, inhibit pathways crucial for cell proliferation and survival, with resistance potential.

11. Conclusion

• Key Idea: Understanding membrane receptors and their signaling cascades, including crosstalk, ligand bias, and the roles of drugs, is critical in cell biology and pharmacology.