CBIO3400 L23 – Signaling via Receptor Tyrosine Kinases (RTKs) #2

CBIO3400 - Signaling via Receptor Tyrosine Kinases (RTKs) - Lecture Notes (04/20/2026)

Classroom Logistics

  • Reminder: Grab an index card on entry for activities and notes.

Practice Exam Question Corrections

  • Cartoon Analysis: Study the cartoon regarding Ca2+-release. Consider the following scenarios:
      a) If you express a GTP that cannot be hydrolyzed:
      - Outcome: Increased Ca2+-release.
      b) If you overexpress IP3 (inositol trisphosphate):
      - Outcome: Increased Ca2+-release.
      c) If the Ca2+-ATPase is mutated:
      - Outcome:
        - ATPase: Increased Ca2+-release.
        - Ca2+-channel: Nothing or decreased Ca2+-release.

Learning Objectives

  1. Explore the Cohen (1982) Experiment: Understanding the methodology and results.
  2. Familiarization with RTK Research Techniques: Bioassays, Western blotting, etc.
  3. Understanding Protein Domains: Their roles in cellular signaling processes.
  4. Intracellular Signaling Complexes: Formation and function of complexes.
  5. In-Class Activity: Engage in hands-on learning (3 points).

Types of Cell-Surface Receptors

  1. Enzyme-Coupled Receptors:
       - Examples: EGF (Epidermal Growth Factor), TGF (Transforming Growth Factor), Insulin, Integrin, FGF (Fibroblast Growth Factor).
       - Functions: Development, cancer, tissue renewal, and functional regulation.
  2. G-Protein-Coupled Receptors (GPCRs):
       - Examples: Serotonin, Dopamine, Acetylcholine.
       - Functions: Relevant in the nervous system; influence behavior, reproduction, and sleep.
  3. Ion-Channel-Coupled Receptors:
       - Functions: Ion transport regulation (like H2O control, membrane potential, ion concentration management influencing the cell cycle and muscle function).

The Discovery of the RTK: EGFR

  • Research Focus: Estranging the role of Epidermal Growth Factor Receptor (EGFR) in signaling pathways.

Freed et al. (2015) Research Insights

  • Utilization of Anti-Phospho-Tyrosine Antibodies: Enabling the detection of phosphorylated form of RTKs post EGF treatment.
  • Evidence obtained via original Western blot data.

Functional Role of EGF Signaling

  • Key Processes Involved:
      - Cell proliferation
      - Cell growth
      - Cell differentiation
  • Various ligands and respective receptors:
      - NRG3, NRG4, NRG1, NRG2, Betacellulin, HB-EGF, Epiregulin, EGF, and TGFα among others.

Activation of hEGFR via Asymmetric Kinase Dimerization

  1. Inactive State: Absence of ligand keeps EGFR in an inactive state.
  2. Ligand Binding: EGF attaches to EGFR, leading to a conformational change prompting dimerization of external domains.
  3. Dimer Formation: This asymmetrical dimerization causes the kinase domain of the activator to exert pressure on the receiver kinase domain.
  4. Phosphorylation Events: The activated receiver domain phosphorylates tyrosines in both domains, leading to the generation of docking sites for intracellular signaling proteins.

Activation of RTKs - Mechanism of Action

  • Phosphorylation: Alters protein conformation, enabling other signaling pathway proteins to bind and become phosphorylated, propagating the signal downstream.

Concerns Regarding Signal Specificity

  • Importance of Signaling Specificity: The interaction relies on protein domains (e.g., SH2 and PTB) which recognize phosphorylated tyrosine residues, which could lead to incorrect signaling if specificity is lost.

Formation of Intracellular Signaling Complexes

  1. Types of Complexes:
      - Type #1: Assembly on activated receptor
      - Type #2: Signaling complex assembly on phosphoinositide docking sites
Summary of Properties of Types #1 and #2
  • Transmission Speed:
      - Type #1: Fast
      - Type #2: Slow (unless kinase concentration is sufficiently high)
  • Signal Amplification:
      - Type #1: Limited amplification opportunities
      - Type #2: High amplification potential
  • Cross-Talk:
      - Type #1: Minimal
      - Type #2: High

Activation of Large Macromolecular Complexes

  • Domain Specificities: Domains like SH2, PTB, and SH3 facilitate specific binding, crucial for effective signaling pathway interactions.

Protein Domains in Signaling

  • Functionality: Proteins often contain multiple domains folded into distinct structures, allowing shuffling to evolve new functions.
  • Example: Protein kinases with kinase domain coupled with recognition domain.

Specific Effectors and Their Binding Sites

  • Key Experiments: Pathway interactions, e.g., PDGF receptor effects on RTKs through various SH2 domains, emphasizing the specificity of these interactions.

In-Class Activity - Mutant Tyrosine Variants of HER1/EGFR

  • Objective: Generate variants to study structural impact on signalling pathways.

Immunoprecipitation Methodology

  • Usage:
      - Procedure: Bind antibodies to magnetic beads for target interaction studies.
      - Reverse lysate to identify direct interactions in proteins through pull-down assays.

RTK Signaling Pathway Examples

  • Highlighted Pathways:
      a) Ras/MAPK Pathway
      b) PI3K/Akt Pathway
      c) Role of Phosphatidylinositol (PIPs) in signaling.

Ras/MAPK Pathway Details

  • Role of Serine/Threonine Kinases: MAPK activation is essential for signaling within the cell.

Conclusion

  • Goal of Signaling Pathways: Regulation of cell proliferation, survival, migration, and gene expression, emphasizing the complexity and necessity for precise communication within cellular environments.