Premed · Premed · Cell Biology
Lecture 8: Cell Signaling II: Receptor Tyrosine Kinases
Cell Biology
Learning Objectives
By the end of this lecture, students will be able to:
- Describe the structure and activation mechanism of receptor tyrosine kinases (RTKs)
- Explain the Ras-MAPK signaling cascade and its downstream effects
- Describe the PI3K-Akt signaling pathway and its role in cell survival
- Explain how RTK signaling is regulated and terminated
- Discuss the role of RTK mutations in cancer
Lecture Content
I. Receptor Tyrosine Kinases — Overview and Structure
Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic tyrosine kinase activity. There are approximately 58 RTKs in humans, organized into about 20 subfamilies. Each RTK has a characteristic architecture: an extracellular domain that binds ligand (containing immunoglobulin-like, cysteine-rich, or fibronectin-type domains), a single transmembrane alpha-helix, and an intracellular domain that houses the tyrosine kinase and regulatory regions.
Major RTK families and their ligands include the EGF receptor family (EGFR/ErbB, activated by EGF and TGF-alpha), the PDGF receptor (activated by PDGF), the FGF receptor (activated by FGF), the VEGF receptor (critical for angiogenesis, activated by VEGF), the insulin receptor (activated by insulin and IGF-1), Trk receptors (activated by neurotrophins such as NGF and BDNF), c-Kit (activated by stem cell factor), and Met (activated by hepatocyte growth factor).
II. RTK Activation Mechanism
RTK activation follows a well-defined sequence. Most RTKs exist as monomers in their inactive state. Ligand-induced dimerization is the critical activating event: ligand binding causes two receptor monomers to come together as a dimer, or in some cases stabilizes a pre-formed dimer. Some ligands, such as PDGF, are themselves dimeric. In the case of EGFR, ligand binding induces a conformational change that exposes a dimerization arm on the extracellular domain.
Dimerization brings the two intracellular kinase domains into close proximity, enabling trans-autophosphorylation, in which each kinase phosphorylates specific tyrosine residues on the opposite receptor. These phosphotyrosine residues then serve as docking sites for downstream signaling proteins that contain SH2 (Src Homology 2) domains or PTB (Phosphotyrosine Binding) domains, each of which recognizes specific phosphotyrosine motifs. Because different phosphotyrosines recruit different proteins, multiple signaling pathways are activated simultaneously from a single receptor. Adaptor proteins such as Grb2, Shc, and Nck have no enzymatic activity but serve as molecular scaffolds. Enzymes recruited to the activated receptor include PLC-gamma, PI3K, Src family kinases, and RasGAP.
The insulin receptor is a notable exception to the general mechanism. It exists as a preformed alpha2-beta2 disulfide-linked dimer. Insulin binding induces a conformational change and trans-autophosphorylation, and the phosphorylated receptor recruits IRS (insulin receptor substrate) proteins rather than the same adaptor set used by other RTKs.
<image>Receptor tyrosine kinase activation mechanism. Panel A: Two inactive RTK monomers in the membrane, each with extracellular ligand-binding domain, single transmembrane helix, and intracellular kinase domain (unphosphorylated). Panel B: Ligand (e.g., EGF) binds, inducing receptor dimerization. Panel C: Trans-autophosphorylation — each kinase domain phosphorylates tyrosine residues on the opposite receptor (shown as yellow "P" circles). Panel D: Phosphotyrosine residues recruit downstream signaling proteins containing SH2 domains (Grb2 shown binding via SH2 domain) and PTB domains, initiating multiple signaling cascades (arrows pointing to Ras-MAPK, PI3K-Akt, and PLC-gamma pathways).</image>
III. The Ras-MAPK Pathway
The Ras-MAPK pathway is one of the most important signaling cascades in cell biology, linking growth factor stimulation to changes in gene expression and cell proliferation. Ras activation begins when the adaptor protein Grb2 binds a phosphotyrosine on the activated RTK via its SH2 domain. Grb2 in turn recruits SOS (Son of Sevenless) through its SH3 domains. SOS is a GEF for Ras, promoting the exchange of GDP for GTP on Ras. Ras-GTP is the active form.
Ras itself is a small monomeric GTPase of the Ras superfamily -- importantly, it is not a heterotrimeric G-protein. Ras is anchored to the inner leaflet of the plasma membrane through a farnesyl lipid modification and acts as a molecular switch, toggling between its active GTP-bound and inactive GDP-bound states. GAPs stimulate GTP hydrolysis to inactivate Ras. RAS mutations are found in approximately 30 percent of all human cancers, with KRAS being the most frequently mutated.
The MAPK cascade propagates the signal from Ras through a three-kinase relay. Ras-GTP recruits Raf (MAPKKK) to the membrane, where Raf becomes activated. Raf phosphorylates and activates MEK (MAPKK), a dual-specificity kinase. MEK phosphorylates ERK (MAPK) on both threonine and tyrosine residues. Activated ERK translocates to the nucleus, where it phosphorylates transcription factors including Elk-1, c-Fos, c-Myc, and c-Jun. These transcription factors drive expression of genes involved in cell proliferation, differentiation, and survival, including cyclin D1, which promotes cell cycle progression. Each step in the cascade amplifies the signal, as each activated kinase phosphorylates many molecules of the next kinase in the sequence. Scaffold proteins such as KSR organize the components of the MAPK cascade for efficient signaling.
IV. The PI3K-Akt Pathway
The PI3K-Akt pathway is a central regulator of cell survival, growth, and metabolism. PI3K (Phosphoinositide 3-Kinase) is recruited to the activated RTK through the SH2 domains of its regulatory subunit (p85). The catalytic subunit (p110) then phosphorylates the membrane lipid PIP2 to generate PIP3, a membrane-bound lipid second messenger.
Akt (Protein Kinase B) contains a PH (Pleckstrin Homology) domain that binds PIP3, recruiting Akt to the membrane, where it is phosphorylated and fully activated by PDK1 and mTORC2. Active Akt phosphorylates a broad array of substrates that collectively promote cell survival by inactivating the pro-apoptotic protein BAD and the pro-apoptotic transcription factors FoxO, and by activating MDM2 to promote p53 degradation. Akt promotes cell growth by activating mTORC1 (through inhibition of the TSC1/TSC2 complex), which in turn stimulates protein synthesis and ribosome biogenesis via S6K and 4E-BP1. Akt drives metabolic changes including GLUT4 translocation to the plasma membrane (the basis of insulin-stimulated glucose uptake) and glycogen synthase activation. It also promotes cell cycle progression by inducing cyclin D1 expression and inhibiting the CDK inhibitor p27.
PTEN is a critically important tumor suppressor phosphatase that opposes PI3K by dephosphorylating PIP3 back to PIP2. Loss of PTEN leads to constitutive Akt activation, and PTEN is one of the most frequently mutated genes in human cancer.
<image>The PI3K-Akt signaling pathway. Panel A: Activated RTK recruits PI3K to the membrane. PI3K phosphorylates PIP2 to generate PIP3 in the inner leaflet. PTEN dephosphorylates PIP3 back to PIP2 (shown as opposing arrows). Panel B: Akt binds PIP3 via its PH domain, is phosphorylated by PDK1 and mTORC2, and becomes fully active. Panel C: Branching arrows from active Akt showing its major downstream effects — (1) Survival: phosphorylation and inactivation of BAD and FoxO, (2) Growth: activation of mTORC1 leading to protein synthesis, (3) Metabolism: GLUT4 translocation and glycogen synthesis, (4) Proliferation: cyclin D1 expression. Each branch includes the relevant substrates and outcomes.</image>
V. Other RTK-Activated Pathways
The PLC-gamma pathway is activated when PLC-gamma is recruited to the phosphorylated RTK via its SH2 domains. Unlike PLC-beta, which is activated by GPCRs through G-alpha-q, PLC-gamma is activated directly by RTKs. However, it cleaves the same substrate (PIP2) to produce the same two second messengers: IP3, which triggers Ca2+ release from the ER, and DAG, which activates PKC.
The JAK-STAT pathway is associated with cytokine receptors rather than classical RTKs. Cytokine receptors lack intrinsic kinase activity but associate with JAK (Janus kinase) family members. Ligand binding activates JAKs, which phosphorylate the receptor and then phosphorylate STAT (Signal Transducer and Activator of Transcription) proteins. Phosphorylated STATs dimerize and translocate to the nucleus to activate gene transcription. This pathway is particularly important in immune cell signaling by interferons and interleukins.
Crosstalk between pathways is pervasive. RTK and GPCR pathways can converge on shared effectors such as Ras and MAPK. GPCR transactivation of EGFR occurs when G-protein signaling activates metalloproteinases that cleave membrane-bound EGF-like ligands, releasing them to activate EGFR. The integration of multiple signals ultimately determines cell fate.
VI. Regulation, Termination, and Clinical Significance
Negative regulation of RTK signaling occurs at multiple levels. Receptors are internalized via clathrin-mediated endocytosis and can be ubiquitinated by the E3 ubiquitin ligase Cbl, targeting them for lysosomal degradation. Protein tyrosine phosphatases (PTPs) remove phosphate groups from tyrosine residues. Sprouty proteins inhibit the Ras-MAPK pathway, and SOCS proteins inhibit the JAK-STAT pathway.
RTK mutations in cancer are among the most clinically important findings in molecular oncology. Gain-of-function alterations that constitutively activate RTK signaling are common drivers of cancer. HER2/ErbB2 overexpression occurs in about 20 percent of breast cancers. EGFR mutations drive many lung adenocarcinomas. The BCR-ABL fusion protein, created by the Philadelphia chromosome translocation, is a constitutively active tyrosine kinase that drives chronic myeloid leukemia (CML). Kit mutations cause gastrointestinal stromal tumors. The BRAF V600E mutation, which constitutively activates Raf, is found in approximately 50 percent of melanomas. RAS mutations, particularly KRAS G12V and G12D, are found in pancreatic, colorectal, and lung cancers.
These molecular insights have led to highly effective targeted cancer therapies. Small molecule inhibitors include imatinib (Gleevec) for BCR-ABL in CML, gefitinib and erlotinib for EGFR in lung cancer, vemurafenib for BRAF V600E in melanoma, and lapatinib as a dual EGFR/HER2 inhibitor. Monoclonal antibodies include trastuzumab (Herceptin) targeting HER2 in breast cancer, cetuximab targeting EGFR in colorectal cancer, and bevacizumab (Avastin) targeting VEGF to block angiogenesis.
