Receptor tyrosine kinases (L4) Flashcards
How are receptor tyrosine kinases classed and how many families are there?
16 families each with many individual receptors - they all have a kinase domain and one transmembrane spanning domain. They are classed by their extracellular domain
What is a kinase insert region?
An extra segment emerging from the folded kinase domain which interrupts the tyrosine kinase domain
Explain canonical RTK activation
The ligand has the ability to dimerise and it facilitates the dimerisation of the receptor. This dimerisation brings together 2 cytoplasmic kinase domains, thereby processing their activation. The kinases phosphorylate each other. This increases the activity of the kinase, stabilises the receptor, causes the kinase to phosphorylate other tyrosines and create other docking sites. Once the protein has bound to the phosphorylated receptor, the signal is transduced via 2 main branches in the RTK signalling pathways : RAS and PI3K
Explain how genetic engineering can be used to analyse the function of RTKs
Using genetic engineering we can generate DNA that encodes for a normal receptor that is mutated in the kinase domain. The DNA is then expressed in an organism at high levels and poisons the endogenous receptor (a dominant negative mutation). We can also generate DNA that encodes for a receptor that lacks a ligand binding domain and instead has a homodimerisation domain. This DNA is then expressed in an organism at normal levels and is ligand-independent (constitutively active)
What are HSPGs?
Heparin sulphate proteoglycans. Multifunctional extracellular matrix components. They have a protein core, which can be transmembrane, tethered or secreted
They have long chains of sugars and each sugar can be modified in many different ways, especially by sulphation. This modification could result in a code that creates binding sites for specific proteins e.g. FGF2. FGF and its receptor form a complex with HSPGs where it first forms oligomeres
Explain the RAS/MAPk pathway
Rasis activated by GTP. Binding of GRB2 and Sos (GEF) couple the receptor to inactivate Ras. SOS promotes dissociation of GDP from Ras: GTP binds and SOS dissociates from active Ras. Mitogen activated protein kinase (MAPK) leads to amplification via a rapid and transient response (a cascade). Active ras activates Map kinase kinase kinase. Map KKK phosphorylates Map KK. Map KK phosphorylates Map K, and then Map K goes on to phosphorylate proteins and regulatory genes to alter protein activity and gene expression
Explain the different methods for studying
Visualisation or detection of interactions/signalling. For in vitro use biochemical methods (columns). For in vivo you can use FRET
Chemical inhibitors to disrupt by manipulating a particular component: agonist Vs antagonist
Misexpression/overexpression
- Wild type Vs constitutively active/ dominant negative.
Genetic methods like knockouts, transgenic, mutation analysis. - google how fret works plz future kirsty
How can you view fluorescently tagged things?
You use a special microscopy camera which contains a laser/mercury lamp and a dichroic mirror to only reflect shorter wavelengths. Colour cameras aren’t sensitive enough so you use greyscale cameras.
