Lec 22 RTKs, Ras/MAPK, and Insulin Signaling

Receptors and Receptor Tyrosine Kinases (RTKs): Classification and Overview

  • Receptors can be classified into several basic categories:
    • Ligand-gated channels
    • Plasma membrane receptors
    • Those linked to G proteins
    • Those linked to protein kinases
    • Nuclear receptors (cytosolic receptors)
  • Receptor Tyrosine Kinases (RTKs) are a major family within plasma membrane receptors that have intrinsic kinase activity and respond to growth factors and other extracellular cues.

RTKs: Structure and Basic Mechanism

  • All RTKs share a common topology:
    • Extracellular ligand-binding domain
    • A single transmembrane domain
    • A cytoplasmic domain with kinase activity
  • Activation mechanism (two key steps):
    • Ligand binding induces receptor dimerization (two mechanisms): the receptor forms a dimer upon ligand binding.
    • Dimerization leads to trans-autophosphorylation of cytoplasmic tyrosine residues (multiple phosphorylation sites).
  • Activated, phosphorylated tyrosine residues serve as docking sites for cytoplasmic signaling proteins that contain SH2 or PTB domains, enabling downstream signaling.
  • Signaling enzymes that commonly associate with RTKs include: protein kinases, protein phosphatases (e.g., Shp2), lipid kinases (PI3K), phospholipases (PLC), and GTPase-activating proteins (GAPs).

Key Molecular Interactions at the Receptor

  • Phosphorylated Tyr residues on RTKs create high-affinity docking sites for SH2-domain proteins (~100 aa SH2 domains) and PTB-domain proteins (~100–150 aa).
  • SH2-domain proteins in humans: ~115, including Grb2 (an adaptor) and many others; adaptor proteins can carry SH2 or PTB domains plus additional protein–protein interaction sites.
  • PTB-domain proteins (e.g., IRS) can also bind RTKs, leading to downstream phosphorylation of PTY sites that act as scaffolds for multiple adaptors or SH2-containing enzymes.
  • Grb2 adaptor protein contains two SH3 domains that bind proline-rich sequences and constitutively associates with Sos (a Ras-GEF) to link RTKs to Ras activation.
  • Adaptor/scaffolding proteins function as linkers to assemble signaling complexes at the membrane, enabling coordinated signaling.

Termination and Re-sensitization of RTK Signaling

  • Termination of RTK signaling occurs via internalization of the receptor (internalization pathways are similar to GPCR termination).
  • RTKs do not require Arrestin for internalization; they have short cytoplasmic motifs that interact with AP2 (clathrin adaptor).
  • Three possible outcomes after endocytosis:
    1) Re-sensitization: receptor returns to the cell surface and can signal again.
    2) Endosomal signaling: receptor signaling continues within endosomes, potentially engaging different pathways.
    3) Degradation: receptor trafficked to lysosome, reducing ligand sensitivity.
  • Receptors can be recycled back to the surface to restore ligand sensitivity.

Ras and the MAPK Pathway: From RTK to Gene Response

  • Many RTKs activate the Ras/MAPK signaling module.
  • Ras is a small monomeric GTPase cycling between active GTP- and inactive GDP-bound states.
    • Regulators:
    • GEFs (e.g., Sos) promote GDP release and GTP binding to activate Ras.
    • GAPs accelerate GTP hydrolysis to inactivate Ras.
    • GDIs inhibit GDP release, keeping Ras in the inactive state.
  • How RTKs activate Ras:
    • Ligand-bound RTKs recruit adaptor proteins (e.g., Grb2) via SH2 domains.
    • Grb2 is bound to Sos via SH3 domains; this complex brings Sos to the membrane where Ras is located.
    • Sos acts as a Ras-GEF to promote the GDP→GTP exchange on Ras, yielding active Ras.
    • Active Ras then engages downstream kinases to propagate the signal.
  • Ras downstream signaling (MAPK cascade):
    • Ras-GTP binds and activates Raf (MAPKKK).
    • Raf phosphorylates and activates MEK (MAPKK).
    • MEK phosphorylates and activates ERK (MAPK).
    • Activated ERK translocates to the nucleus and phosphorylates transcription factors such as Ets and c-Jun, driving gene expression and cell proliferation.
  • Key experimental evidence that Ras functions downstream of RTKs:
    • PDGF and EGF induce cell proliferation; microinjection of anti-Ras antibodies blocks proliferation.
    • Dominant-negative Ras inhibits proliferation; constitutively activated Ras causes proliferation without external signals.
    • Constitutively active Ras mutations (e.g., at position 12 in RasD; G12 mutations) prevent GAP-mediated inactivation, keeping Ras in the GTP-bound active state.
    • About ~30% of human tumors harbor hyperactive Ras signaling.
  • Drosophila eye as a model system to study RTK signaling components:
    • The eye has ~800 ommatidia; R8 photoreceptors signal to specify R7 fate.
    • Sevenless ( Sev ) is a receptor tyrosine kinase expressed in R8; Boss is its ligand (a seven-pass transmembrane receptor expressed on the surface of R8) that activates Sev.
    • Sev activation recruits Drk (the Drosophila Grb2 homolog) and Sos to activate Ras downstream, leading to R7 specification.
    • Genetic screens using a temperature-sensitive Sev mutation helped identify downstream components that function in Sev signaling; many downstream components are essential and lethal when mutated, complicating screens.
  • Drosophila Sev pathway details:
    • Boss (ligand) binds Sev (RTK) on the R8 cell.
    • Grb2 homolog Drk and Sos connect Sev to Ras, activating Ras.
    • Ras activation then triggers the MAPK cascade similar to vertebrates, leading to specification of the R7 photoreceptor.
    • Ras acts downstream of Sev to transmit the signal for R7 induction.
  • The Ras→MAPK cascade and cancer relevance:
    • Activated Ras-GTP binds RAF (MAPKKK) at the membrane, initiating the cascade that leads to proliferation signals.
    • Ras must be inactivated to prevent perpetual signaling; RasGAPs accelerate GTP hydrolysis to shut off the signal.
    • Ras mutations that lock Ras in the GTP-bound state contribute to oncogenesis by constitutively activating the MAPK pathway.

MAPK Cascade Specificity and Scaffolding

  • Specificity in MAPK signaling is achieved by spatial localization and by scaffolding proteins that tether specific pathway components together.
  • Different MAPKKKs can phosphorylate distinct MAPKKs, which in turn phosphorylate specific MAPKs, leading to different cellular outcomes (e.g., cell cycle progression vs. arrest).
  • Scaffolding molecules can link signaling components and confine signaling to a particular time and place, reducing cross-talk.
    • Yeast example: Ste5 (MAPKKK scaffold) and Pbs2 (MAPKK scaffold) can redirect a mating pathway signal to osmoregulatory signaling when combined, illustrating control of signaling outcomes by scaffolds.
  • AKAPs illustrate scaffolding in cAMP signaling and show how scaffolds can cluster kinases for localized responses.
  • GPCRs and RTKs differ in their wiring, but both can engage MAPK modules via different adaptor and scaffold strategies to yield specific cellular responses.

Insulin Receptor Signaling and the PI3K-AKT Pathway

  • The insulin receptor is an RTK; upon activation, insulin receptor substrates (IRS) are phosphorylated on tyrosines, creating docking sites for SH2-domain proteins.
  • IRS phosphotyrosines recruit SH2-domain-containing signaling proteins, propagating the signal through the PI3K pathway and beyond.
  • Phosphoinositide 3-kinase (PI3K) role:
    • Converts PI(4,5)P2 to PI(3,4,5)P3 (PIP3) at the inner leaflet of the plasma membrane:
      extPI(4,5)P<em>2ightarrowextPI(3,4,5)P</em>3(PIP3ext)ext{PI(4,5)P}<em>2 ightarrow ext{PI(3,4,5)P}</em>3 \text{(PIP}_3 ext{)}
  • PIP3 serves as a docking site for PH-domain containing kinases, notably PKB/AKT and PDK1, bringing them to the membrane for activation.
  • PH domains (Pleckstrin Homology domains) are ~120 amino acids and bind distinct phosphoinositides in membranes, enabling recruitment of signaling proteins to PIP3-rich regions.
  • Activation of AKT (PKB) pathway:
    • AKT is recruited to the membrane via its PH domain binding PIP3.
    • AKT is phosphorylated and activated by PDK1 (and mTORC2 provides a second phosphorylation) to become fully active.
    • Active AKT phosphorylates multiple targets to promote glucose uptake and cell growth, including inhibition of GSK3 and promotion of glucose metabolism.
  • Consequences for glucose uptake and glycogen synthesis:
    • AKT signaling promotes translocation of the glucose transporter GLUT4 to the plasma membrane, increasing glucose uptake.
    • AKT also inhibits negative regulators of glycogen synthesis, notably by inactivating GSK3, thereby allowing glycogen synthase activity to increase.
  • Regulation and feedback:
    • PTEN (a lipid phosphatase) opposes PI3K by dephosphorylating PIP3 to PI(4,5)P2, acting as a brake on the pathway.
  • Practical significance:
    • Insulin signaling via PI3K-AKT links extracellular insulin levels to glucose uptake and storage, tying signaling to metabolic control.

Protein Tyrosine Phosphorylation in Context

  • Section focus: 15.5 – Protein-Tyrosine Phosphorylation; understanding how Tyr phosphorylation coordinates RTK signaling, adaptor proteins, and downstream kinases.
  • Compare RTK signaling strategies to other receptor families (e.g., GPCRs) to appreciate distinctive features such as dimerization-induced activation, SH2/PTB docking, and robust Ras/MAPK signaling.

Key Domains, Adapter Proteins, and Their Roles

  • SH2 domains: ~100 aa; bind phosphotyrosine-containing motifs on activated receptors and docking proteins; ~115 SH2-domain-containing proteins in the human genome.
  • PTB domains: ~100–150 aa; bind phosphotyrosines in certain contexts (often within NPXpY motifs) and can mediate alternative docking to RTKs (e.g., IRS).
  • Grb2: adaptor with SH2 domain to bind pY sites on RTKs and two SH3 domains that recruit Sos (Ras-GEF) via polyproline interactions.
  • Sos (Son of Sevenless): Ras-GEF that catalyzes GDP release to enable Ras activation.
  • IRS (Insulin Receptor Substrate): PTB-domain-containing docking protein that binds RTKs and recruits SH2-domain signaling proteins to propagate insulin signaling.
  • Adaptor/scaffolding proteins: enable formation of multi-protein signaling complexes; can contain SH2 or PTB and other interaction motifs; critical for signal specificity and efficiency.

The Insulin Receptor: A Case Study of RTK Signaling

  • The receptor itself is an RTK with disulfide bonds that create high-affinity docking sites for SH2/PTB-containing molecules.
  • IRS docking sites with SH2 domains recruit PI3K and other signaling proteins, coupling insulin binding to metabolic responses.
  • This system highlights how tyrosine phosphorylation organizes metabolic signaling beyond cell growth control.

Experimental Takeaways and Exam-Relevant Points

  • Ras functions downstream of RTKs; Ras must cycle between GTP- and GDP-bound forms, regulated by GEFs (activate) and GAPs (inactivate).
  • Ras mutations can cause constitutive MAPK signaling and contribute to tumorigenesis; roughly 30% of human tumors harbor hyperactive Ras variants.
  • The Drosophila Sev-Boss system demonstrates a real organismal example of RTK signaling components and how downstream elements (Drk, Sos, Ras) drive cell fate decisions (R7 specification).
  • Specificity in MAPK signaling is achieved via scaffold proteins and spatial localization, ensuring that the same core module can elicit different cellular responses.
  • The insulin PI3K-AKT pathway translates extracellular insulin into intracellular metabolic actions, including GLUT4 translocation and glycogen synthesis through AKT-mediated regulation of GSK3 and glycogen synthase.

Summary: What You Should Know by the End

  • The general strategy of RTK signaling vs. GPCR signaling, including ligand-induced receptor dimerization and trans-autophosphorylation.
  • The role of PTB and SH2 motifs in docking adaptors and effectors to RTKs.
  • The downstream kinase cascade activated by RTKs, with MAPK as a central module.
  • The difference between heterotrimeric G proteins and small monomeric GTPases like Ras, including their cycles of activation and inactivation (GEFs, GAPs, GDIs).
  • The role of adaptor/scaffold proteins in assembling signaling complexes and conferring specificity.
  • A model organism example (Drosophila Sevenless) illustrating how RTK signaling components (Boss, Sev, Drk, Sos, Ras) drive development.
  • The mechanism of insulin receptor signaling through IRS and the PI3K–AKT pathway, including PIP3 docking and AKT activation, leading to glucose uptake and glycogen storage.
  • The concept of signaling termination and receptor trafficking: internalization, endosomal signaling, degradation, and recycling.
  • The significance of signaling specificity via scaffolding and compartmentalization, and how improper regulation can contribute to disease (e.g., cancer).