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Flashcards in Lecture 20 Deck (43)
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What can be said about the transmembrane domain in RTKs

The transmembrane domain is said to lack structure and be very simple. It is short and string like, consisting of between 25 and 38 amino acid residues


How long after RTK activation is gene transcription influence

Within minutes


Other than Heparan Sulphate, give some examples of sugar side chains present on proteoglycans

Aggrecan, Betaglycan, Decorin and Perlecan


What is the result of stimulation of the TrkA receptor by binding of NGF

Stimulation of survival and growth of some neurons


SH2 domains that bind to phosphotyrosines in RTKs also recognise adjacent residues. What is the recognition sequence which they recognise



Explain how Ras acts as a molecular switch in downstream RTK signalling

Ras is a smallGTPase that functions as a molecular switch. Its nucleotide-binding site is formed by several protein loops that cluster one end of the protein. Inactive Ras is bound tightly to GDP which is displaced by GTP when Ras becomes active. Ras can toggle between two conformational states depending on whether GTP or GDP is bound. The switch 1 and switch 2 regions change conformation dramatically between to two Ras states and this conformational change allows other proteins to distinguish activate Ras from inactive Ras. Active Ras binds to and activates, downstream target proteins in the cell signalling pathways. Hydrolysis of GTP to inactivate Ras requires the action of Ras-GAP which binds tightly to Ras burying the bound GTP. Ras-GAP inserts an arginine side chain directly into the active site. This Inserted arginine with threonine and glutamine side chains of Ras itself, promotes the hydrolysis of GTP.


HSPGs can also be entirely secreted, T or F



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. Similarly, ligand binding may also reorientate existing receptor oligomers. 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.


What species attached to the proteoglycan backbones can be sulphated to trigger ligand binding to the FGF receptor



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


What is the result of stimulating the EGF receptor tyrosine kinase

Stimulation of proliferation


Why is a greyscale camera used to study fluorescence

Colour cameras aren’t sensitive enough to produce high quality images. Instead, colour is added to the greyscale image later to create what is known as pseudocolour


What can be said about the extracellular domains of RTKs throughout the family

The extracellular domains vary greatly along with the ligands. They do however share features such as Ig-like and fibronectin-like domains and often contain several repeating units


What is the result of stimulating the IGF receptor

Stimulation of carbohydrate utilisation and protein synthesis


Explain how the apparatus for fluorescent microscopy works

In addition to the light on the specimen, fluorescent microscopes also have a light of a specific wavelength shone onto the stage. This is either provided by a mercury lamp or a laser. The excitation filter of the microscope is then specific for the fluorophore used to stain particular regions of the specimen. A dichroic beam splitter mirror is used to reflect only short wavelengths hence allowing longer wavelengths to pass straight through. As fluorescent objects emit light of a longer wavelength than was shone onto it, the emitted light from the specimen passes through into the eyepiece. A greyscale camera then measures the light intensity and creates a greyscale image which can later be analysed


What phenomenon is FRET said to rely on

Paired fluorescence


Explain the role of HSPGs in FGF signalling

FGF and its receptor forms a complex with heparan sulphate proteoglycans. HSPGs provide an extracellular scaffold for FGF and presents it to the receptor after it has oligomerised on the HSPG


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


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


Cyclins are an example of downstream targets of MAP-Kinases, T or F



Modification by sulphation of GAGs can provide a code which creates binding sites for specific proteins and sequences that carry information, T or F



Give some examples of HSPGs

Glypican, Syndecan and Perlecan


HSPGs can be membrane tethered in two ways. Describe these

HSPGs can be tethered to the cell membranes either by a transmembrane domain within the proteoglycan backbone or through a lipid modification such as GPI anchors


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


What sorts of cell behaviours are RTKs involved in regulating

Proliferation, differentiation and migration


What component of the extracellular matrix do RTK ligands often form complexes with

Heparan sulphate proteoglycans (HSPGs)


The pattern of sulphation acts as a code allowing specific HSPGs to interact with specific proteins, T or F



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


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 at high levels by incorporation of a transgene will result 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. By expressing this transgene at high levels there is no need to interfere with the other endogenous gene.


Most RTKs are monomers with one major exception, which receptor is this

The insulin receptor is an RTK which is present as a dimer