Lecture 29 – Receptor Protein-Tyrosine Kinases

Q: What type of signalling pathway do receptor tyrosine kinases (RTKs) use?

RTKs use a phosphorylation cascade, though some proteins involved can also participate in second messenger pathways.

Q: What is the difference between receptor tyrosine kinases and cytoplasmic protein-tyrosine kinases?

RTKs are directly activated by extracellular signals and have a ligand-binding domain, while cytoplasmic protein-tyrosine kinases are purely cytoplasmic, not receptor-bound, and are regulated indirectly by extracellular signals (e.g., Jak2).

Q: What is ligand-mediated dimerization?

One ligand has two receptor-binding sites and acts like a dumbbell, binding two separate receptor chains and bringing them together. PDGF is an example.

Q: What is receptor-mediated dimerization?

Each receptor chain binds one ligand, and two ligand-bound receptors then dimerize. EGF is an example.

Q: What happens after RTK dimerization?

Dimerization brings two kinase domains into close contact, allowing trans-autophosphorylation, which increases kinase activity.

Q: What is trans-autophosphorylation?

It is the process by which dimerized receptor subunits phosphorylate each other on tyrosine residues. It both increases kinase activity and creates docking sites for signalling molecules.

Q: What is the activation loop, and what role does it play in RTK activation?

The activation loop is a region in the kinase domain that, upon phosphorylation, moves away from the substrate-binding site, thereby activating the kinase domain.

Q: What does autophosphorylation on regions adjacent to the kinase domain accomplish?

It creates binding sites (docking sites) for cellular signalling proteins that carry SH2 or PTB domains.

Q: What is an SH2 domain?

An SH2 (Src homology 2) domain binds phosphorylated tyrosine residues. Examples of proteins with SH2 domains include STAT molecules, Grb2, PI3K, Shp2, and PLC.

Q: What is an SH3 domain?

An SH3 (Src homology 3) domain binds hydrophobic proline residues and is not directly dependent on phosphorylation.

Q: What is a PTB domain?

A PTB (phosphotyrosine-binding) domain binds phosphorylated tyrosines, similar to an SH2 domain, but evolved independently.

Q: What is Grb2 and what does it do?

Grb2 is an adaptor protein with an SH2 domain on one side (to bind the receptor) and an SH3 domain on the other (to recruit other proteins such as SOS), linking the activated receptor to downstream signalling.

Q: What is IRS and what is its function?

IRS (insulin receptor substrate) is a docking protein that provides additional phosphorylation sites and extra versatility. It contains a PTB domain to bind the receptor and, once phosphorylated, provides extra docking sites for proteins like PI3K and Shp2.

Q: How do STAT proteins function in signalling?

STATs are both signal transducers (relaying the signal) and transcription factors (binding DNA in the nucleus). They use SH2 domains to bind to the receptor and to each other, and must be tyrosine-phosphorylated to be activated.

Q: How is STAT signalling shut off?

Cytokine-inducible SH2-containing proteins bind to the receptor and prevent STAT docking, blocking further signal transduction.

Q: What are the four categories of signalling proteins that dock to RTKs?

Adaptor proteins (e.g., Grb2), docking proteins (e.g., IRS), transcription factors (e.g., STATs), and signalling enzymes (e.g., protein kinases, lipid kinases, phosphatases, GTPase-activating proteins, phospholipase-C).

Q: What is a GEF and what does it do?

A GEF (guanine nucleotide-exchange factor) stimulates the dissociation of bound GDP, promoting GTP binding and activation of the G protein. It turns the G protein switch ON.

Q: What is a GAP and what does it do?

A GAP (GTPase-activating protein) stimulates hydrolysis of bound GTP by the G protein, decreasing the duration of the signal. It turns the G protein switch OFF.

Q: What is a GDI and what does it do?

A GDI (guanine nucleotide-dissociation inhibitor) inhibits the release of bound GDP, maintaining the inactive GDP-bound state of the G protein. It keeps the G protein switch OFF.

Q: What is Ras?

Ras is a small monomeric G protein that acts as a molecular switch in signal transduction. Unlike heterotrimeric G proteins, small G proteins like Ras need accessory proteins (GEFs, GAPs, GDIs) to regulate each step of their cycle.

Q: What is SOS and how does it relate to Ras?

SOS (Son of Sevenless) is a GEF for the Ras G protein. It helps Ras release GDP and bind GTP, activating it. SOS is recruited to the membrane by the adaptor protein Grb2.

Q: What is the MAP kinase cascade?

The MAP (mitogen-activated protein) kinase cascade is a series of sequential phosphorylation events (MAPKKK phosphorylates MAPKK, which phosphorylates MAPK) that relay signals from activated Ras to transcription factors, regulating cell proliferation and differentiation.

Q: What is a mitogen?

A mitogen is a growth factor that induces cell proliferation and cell division.

Q: What happens at the end of the MAP kinase cascade?

ERKs (MAPKs) activate transcription factors that drive transcription of genes like Cyclin D1, promoting DNA synthesis and progression from G1 to S phase of the cell cycle.

Q: How is the MAP kinase cascade turned off?

Phosphatases are upregulated and dephosphorylate MAPKs. Control also occurs at the Ras level through GAPs that stimulate GTP hydrolysis, inactivating Ras.

Q: What is cross-talk in signal transduction?

Cross-talk occurs when components of one signalling pathway interact with or influence another pathway. For example, signalling enzymes docked to an RTK (like phospholipase-C) can produce second messengers, linking the phosphorylation cascade to second messenger pathways.