Lecture 17 Flashcards
How long after RTK activation is gene transcription influence
Within minutes
Explain the loss of function approach that can be used to investigate RTK signalling
Genetically engineer DNA to generate a gene encoding an RTK whose intracellular kinase domain is mutated. This will lead to a loss of kinase activity and thus no auto and crossphosphorylation. Hence the RTK will be unable to activate in response to ligand binding. This DNA can then be expressed at high levels to result in a dominant negative or antimorphic mutation whereby the mutant RTK will poison the endogenous receptor
What is the overall function of RTKs
Receptor tyrosine kinases phosphorylate tyrosine residues in target proteins
Modification by sulphation of GAGs can provide a code which creates binding sites for specific proteins and sequences that carry information, T or F
T
Which subgroup of FGF receptor ligands are the largest
Paracrine FGFs
How does the MAP kinase pathway rely the signal transduction further from activation of the Ras GTPase
Activated Ras phosphorylates MAP-KKK which then binds to and activates MAP-KK by phosphorylation. Activated MAP-KK then goes onto phosphorylate and activate MAP-K. Activated MAP-Kinase can then phosphorylate transcription factors and other proteins leading to the regulation of gene transcription
What are the effects of auto and cross-phosphorylation of the active RTK
Increased kinase domain activity, stabilisation of the receptor active state (ligand independent) and the creation of docking sites for target proteins
What species attached to the proteoglycan backbones can be sulphated to trigger ligand binding to the FGF receptor
Glycosaminoglycans
Proteins that bind to phosphotyrosines in RTKs by particular domains also recognise adjacent residues. What is the recognition sequence which they recognise
Phosphotyrosine-Glutamate-Glutamate-Isoleucine
There are only 4 different genes for all of the FGF receptors. How is it then that these genes can account for 48 different receptors
The four FGF receptor genes have different splice variants that creates the 48 different isoforms of FGF receptors.
Give some examples of RTK ligands
Ephrins, Nerve Growth Factor, Fibroblast Growth Factor, Epidermal Growth Factor
Give example of roles that FGF signalling plays in development
FGF8 is involved in formation of the limbs. It is also expressed in the somites particularly, in the myotome. FGF8 also plays a part in midbrain-hindbrain patterning
What change in the intracellular environment is a common theme in signal transduction
Changes in the subcellular localisation of components leads to activation of the pathway
Each ligand receptor pair involves one specific ligand and one unique receptor, T or F
F – whilst some ligands are specific for one receptor and vice-versa, some ligands and receptors can be promiscuous and bind to various other components
Cyclins are an example of downstream targets of MAP-Kinases, T or F
T
Explain the role of dimerisation in RTK activation
The RTK ligand can bind as a ligand leading to recruitment of the other receptor monomer. However, this dimerisation of the receptor monomers is the essential stage in RTK activation
Describe the effects of HSPGs on the gradients of secreted molecules
HSPGs control the steepness of a secreted molecule gradient and how far a growth factor can diffuse through the extracellular space
HSPGs are important extracellular modifiers of cell-cell signalling, what is their role in the extracellular environment
They are important in organising the extracellular matrix into basal lamina
The ligands for RTKs all tend to be extracellular, T or F
T
What can be said about the extracellular domains of RTKs throughout the family
The EEC domains vary greatly along with the ligands. They do however share features such as Ig-like and fibronectin-like domains
Proteins containing what type of domain can bind to the phosphorylated tyrosine residues in activated RTKs
SH2 domain containing proteins
What is the role of the acid box domain of the FGF receptor
The acid box domain is involved in negative regulation of the receptor by preventing its activation in the absence of ligand binding
Give some examples or proteins that bind to RTKs
GTPase-activating proteins (GAPs), phospholipase C-? and PI-3 kinases
What can be said about the transmembrane domain in RTKs
The transmembrane domain is short and string like, consisting of between 25 and 38 amino acid residues
What class of receptors are the receptor tyrosine kinases
Enzyme-linked receptors
What are the mammalian homologues of MAP-KKK, MAK-KK and MAP-K
MAP-KKK –> Raf, MAP-KK –> Mek and MAP-K –> Erk
Most RTKs are monomers with one major exception, which receptor is this
The insulin receptor is an RTK which is present as a dimer
What sort of concentration levels do RTK ligands act at
Very low concentrations in the nM or pM range
What component of the extracellular matrix does the FGF receptor-ligand often form complexes with
Heparan sulphate proteoglycans (HSPGs)
Describe the structure of HSPGs
Consist of a proteoglycan core with glycosaminoglycan side chains
MAP-K is regulated by its phosphorylation by MAP-KK, describe how MAP-K is activated
In order to be activated MAP-K must have both of its phosphorylation sites on threonine and tyrosine residues phosphorylated by MAP-KK. These amino acids are interspaced by only one residue and lie in close proximity.
Give some examples of HSPGs
Glypican, Syndecan and Perlecan
What sorts of cell behaviours are RTKs involved in regulating
Proliferation, differentiation and migration
There are 20 different families of RTKs, how many human RTK genes have been identified
58
The protein core of HSPGs tethers them to the membrane, T or F
F – whilst the protein core is the unit responsible for membrane tethering, this doesn’t occur always. Some protein cores are transmembrane domains or can direct the HSPG for secretion
Other than Heparan Sulphate, give some examples of sugar side chains present on proteoglycans
Aggrecan, Betaglycan, Decorin and Perlecan
Outline canonical RTK activation
Following ligand binding, either as a dimer or monomer, the monomeric RTK receptor will dimerise by recruitment of the other receptor monomer. Activation of the RTK causes a change in conformation of the receptor dimer. This starts with the extracellular and transmembrane domains and is then translated to the intracellular kinase domain. This change in conformation of the intracellular domain unmasks the tyrosine kinase domain and exposes important residues for this process. The activated receptor then undergoes auto and crossphosphorylation. This increases the activity of the kinase domains, stabilises the active state of the receptor and causes the kinase domain to phosphorylate other tyrosines in the receptor to create docking sites. These kinase domains are now able to phosphorylate target proteins that bind to the docking site to transduce the signal.
Explain the gain of function approach that can be used to investigate RTK signalling
Genetically engineer DNA to generate a gene encoding an RTK whose extracellular ligand binding domain has been replaced with a homodimerization domain. Expression of this gene in an organism by incorporation of a transgene will results in the production of an RTK capable of dimerising in the absence of ligand binding. This receptor tyrosine kinase will be activated independently of the ligand and known as constitutively active
Describe the structure of the intracellular domain of RTKs
The intracellular domains possess the kinase activity. These are present as a single domain or split into two
Which domains are the ligand binding domains of the FGF receptor
D2 and D3 domains
Explain how activation of RTKs leads to signal transduction by the Ras pathway
Ras is a smallGTPase that is present in the membrane of the cell. The activated RTK contains phosphorylated tyrosine residues in its intracellular kinase domain that have occurred because of autophosphorylation. These phosphotyrosines are recognised by proteins that contain an SH2 domain. In the Ras pathway, this protein is Gbr2 which binds to the activated receptor by its SH2 domain. Gbr2 then recruits another protein to the complex called sos by its SH3 protein-protein interaction domain. Sos is a guanine nucleotide exchange factor (GEF) that is bound to the Ras GTPase. Binding of Gbr2 to sos couples the activated RTK to the inactive Ras. Sos also promotes dissociation of GDP from Ras which is displaced by GTP. Now that it’s bound to GTP Ras dissociates from sos and phosphorylates MAP-KKK to transduce the signal further.
One member of the superfamily of RTKs is the FGF receptor family. What are the three different subgroups of FGF ligands
Paracrine, Endocrine and Intracrine
The FGF receptor-ligand complex can become activated in the absence of binding to components of the extracellular matrix, T or F
F – the receptor can only become activated when in a complex with HSPGs
Below is a list of several proteins involved in signal transduction and RTK activation, determine if they are a GTPases, GEFs, Kinases, GAPs or adapter proteins. Sos, Gbr2, Ras, MAP-KKK, PI-3, Erk, Mek and Raf
Sos - GEF, Gbr2 - Adapter Protein, Ras - GTPase and MAP-KKK, PI-3, Erk, Mek and Raf - kinases
Give an example of a disease caused by mutations in FGF signalling and describe how this accounts for the symptoms
Achondroplasia is a dwarfism disease caused by an activation mutation in the transmembrane domain of the FGF receptor. This means that the receptor is constitutively active independent of ligand binding. FGF signalling then leads to a repression of Ihh which in turn leads to a loss of expression of PTHrP. PTHrP is required to stimulate proliferation and chondrogenesis and hence increased FGF signalling leads to the decreased bone growth characteristic of dwarfism.
What is the effect of having multiple stages in the MAP Kinase pathway
One activated MAP-KKK can phosphorylate and activate several MAP-KK proteins which in turn can phosphorylate and activated multiple MAP-K proteins. This acts as an amplification step in signal transduction
