Receptors with intrinsic enzymatic activity

Question 1

Receptor Tyrosine Kinases (RTKs)

Answer 1

Found in all multicellular organisms
Comprise of a ligand binding domain, one transmembrane helix and a cytoplasmic domain.
Protein kinase activity in the cytoplasmic domain
Kinase activity of domain is stimulated by binding of ligands to the extrace domain.
Ligands are polypeptides

Question 2

Principles of RTK signal transduction

Answer 2

Inactive - no ligand, no tyrosine kinase activity
Ligand binding promotes receptor dimerisation (induced conformational change). This has two effects -
1. Allows Mg-ATP to bind to each tyrosine kinase domain.
2. Allows one of the ATP-bound tyrosine kinases to phosphorylate tyrosines on the other partner in the dimeric complex (transphosphorylation) activating RTK.
Phosphorylated tyrosines act as docking sites for intracellular proteins which relay and amplify downstream signalling.

Question 3

How do RTKs dimerise?

Answer 3

Three ways

1. A monomeric peptide binds to its receptor, inducing conformational changes that promote receptor cross-linking to either another ligand-bound subclass of receptor family or a constituitively active subclass of receptor family. (example is EGF binding to HER1)
2. A preformed dimer (ie has 2 receptor binding sites on separate subunits of molecule) is self crosslinked (example is PDGF)
3. Receptors are expressed as disulphide bond linked preformed heteromers (two units with 2 binding sites on each - one alpha one beta). Ligand binds to alpha binding sites inducing a conformational change that is trasmitted to the beta binding sites which then phosphorylate each other

Question 4

RTK signalling complexes

Answer 4

Approx 20-30 downstream signalling molecules can bind simultaneously to one active dimer. Exact number and composition varies between receptors.
Components include - phosphilpases (hydrolysing PIP2 to DAG + IP3), lipid kinases (PI-3 kinase), and guanine nucleotide exchange factors (GEFs) for small G-proteins (Ras and Rac).
Complexes are disassembled by RTK dephosphorylation by protein tyrosine phosphatases, terminating the signal.

Question 5

How does receptor phosphorylation rely on intracellular signalling?

Answer 5

Phosphorylated tyrosines serve as docking sites for SH2 and PTB domains present on specific intracellular signalling molecules.

Question 6

Ras

Answer 6

Part of GTPase family that controls many celular processes.
Cycles between GDP and GTP bound states
Intrinsic GTPase activity
Permanently active Ras mutations found in many forms of cancer.
Ras controls the MAP kinase pathway (proliferation) and the PI-3 kinase pathways (survival and migration)
Permanently active Ras stimulates these pathways.

Question 7

Inactive Ras

Answer 7

In a GDP-bound off state at the plasma membrane.
Phosphorylated tyrosines acts as docking sites for SH2 domain on an adapter protein Grb2. Grb2 interacts with a Ras guanine nucleotide exchange factor SOS. Membrane localised SOS promotes the release of GDP allowing GTP to bind to Ras, activating it.

Question 8

GTP-Ras (active)

Answer 8

Activates a kinase cascade to turn on the MAP kinase signalling pathway (1) and the PI-3 kinase lipid signalling pathway (2).

(1) Gtp bound Ras recruits a MAP-kinase-kinase-kinase called Raf to the plasma membrane. Raf phosphorylates activating a MAP-kinase-kinase called MEK. MEK phosphorylates activating MAP-kinase. MAP-kinase phosphorylates and alters the actigity of multiple enzymes and trasncription factor to stimulate cell proliferation.
(2) phosphorylated receptor and GTP-Ras recruits PI-3-kinase to the membrane. P85 (regulatory subunit) binds phosphorylated tyrosine and Ras. P110 (a catalytic subunit) phosphorylates membrane lipid PIP2 to PIP3. PIP3 acts as a second messenger, binds and activates PKB. PKB phosphorylates and alters the activity of multiple enzymes and transcriotion factors to stimulate cell survival.

Question 9

How can distinct phosphorylated tyrosine residues interact with specific SH2 domains?

Answer 9

Phosphorylated tyrosine binding site is common to all SH2 domains. Binding site for side chain amino acids is 3 positions down from P-tyr. It is variable between SH2 domains from different proteins thus confers specificity.

Question 10

Uncontroled activation of RTK signalling

Answer 10

Linked with development of cancer
Defined by cells displayed uncontrolled proliferation, inability to differentiate, invasiveness, capacity to metastasize.

Question 11

Zelboraf (vemurafenib)

Answer 11

An ATP binding site inhibitor, orally active, selectively inhibits Val600Glu B-Raf. Recently approved for the treatment of metastatic melanoma.