Lecture 20

Question 1

What can be said about the transmembrane domain in RTKs

Answer 1

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

Question 2

How long after RTK activation is gene transcription influence

Answer 2

Within minutes

Question 3

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

Answer 3

Aggrecan, Betaglycan, Decorin and Perlecan

Question 4

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

Answer 4

Stimulation of survival and growth of some neurons

Question 5

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

Answer 5

Phosphotyrosine-Glutamate-Glutamate-Isoleucine

Question 6

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

Answer 6

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.

Question 7

HSPGs can also be entirely secreted, T or F

Answer 7

T

Question 8

Outline canonical RTK activation

Answer 8

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.

Question 9

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

Answer 9

Glycosaminoglycans

Question 10

The FGF receptor-ligand complex can become activated in the absence of binding to components of the extracellular matrix, T or F

Answer 10

F – the receptor can only become activated when in a complex with HSPGs

Question 11

What is the result of stimulating the EGF receptor tyrosine kinase

Answer 11

Stimulation of proliferation

Question 12

Why is a greyscale camera used to study fluorescence

Answer 12

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

Question 13

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

Answer 13

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

Question 14

What is the result of stimulating the IGF receptor

Answer 14

Stimulation of carbohydrate utilisation and protein synthesis

Question 15

Explain how the apparatus for fluorescent microscopy works

Answer 15

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

Question 16

What phenomenon is FRET said to rely on

Answer 16

Paired fluorescence

Question 17

Explain the role of HSPGs in FGF signalling

Answer 17

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

Question 18

What are the effects of auto and cross-phosphorylation of the active RTK

Answer 18

Increased kinase domain activity, stabilisation of the receptor active state (ligand independent) and the creation of docking sites for target proteins

Question 19

Describe the effects of HSPGs on the gradients of secreted molecules

Answer 19

HSPGs control the steepness of a secreted molecule gradient and how far a growth factor can diffuse through the extracellular space

Question 20

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

Answer 20

T

Question 21

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

Answer 21

T

Question 22

Give some examples of HSPGs

Answer 22

Glypican, Syndecan and Perlecan

Question 23

HSPGs can be membrane tethered in two ways. Describe these

Answer 23

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

Question 24

Describe the structure of the intracellular domain of RTKs

Answer 24

The intracellular domains possess the kinase activity. These are present as a single domain or split into two

Question 25

What sorts of cell behaviours are RTKs involved in regulating

Answer 25

Proliferation, differentiation and migration

Question 26

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

Answer 26

Heparan sulphate proteoglycans (HSPGs)

Question 27

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

Answer 27

T

Question 28

HSPGs are important extracellular modifiers of cell-cell signalling, what is their role in the extracellular environment

Answer 28

They are important in organising the extracellular matrix into basal lamina

Question 29

Explain the gain of function approach that can be used to investigate RTK signalling

Answer 29

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.

Question 30

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

Answer 30

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

Question 31

Give some examples of RTK ligands

Answer 31

Ephrins, Nerve Growth Factor, Fibroblast Growth Factor, Epidermal Growth Factor

Question 32

Give some examples or proteins that bind to RTKs

Answer 32

GTPase-activating proteins (GAPs) that function in the Ras/MAP kinase pathway, phospholipase C-? (part of the inositol lipid pathway) and PI-3 kinases that act as regulatory subunits

Question 33

What are the mammalian homologues of MAP-KKK, MAK-KK and MAP-K

Answer 33

MAP-KKK –> Raf, MAP-KK –> Mek and MAP-K –> Erk

Question 34

Explain the loss of function approach that can be used to investigate RTK signalling

Answer 34

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

Question 35

What is the effect of having multiple stages in the MAP Kinase pathway

Answer 35

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

Question 36

What class of receptors are the receptor tyrosine kinases

Answer 36

Enzyme-linked receptors

Question 37

How does the MAP kinase pathway rely the signal transduction further from activation of the Ras GTPase

Answer 37

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

Question 38

How many different families of RTKs are there

Answer 38

16 different families

Question 39

MAP-K is regulated by its phosphorylation by MAP-KK, describe how MAP-K is activated

Answer 39

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.

Question 40

Explain how fluorescent resonance energy transfer can be used to investigate protein interactions

Answer 40

Recombinant fusion of proteins X and Y to separate fluorescent proteins that absorb and emit certain wavelengths of light allows you to determine if X and Y interact/bind. By correlating the wavelength emitted by the fluorescent protein attached to X with the wavelength of light needed for fluorescence of protein Y you can activate protein Y fluorescence if it is in close proximity to X (i.e. it is bound). I.e. if shining light needed for fluorescence in protein X leads to the appearance of light that is given off as a result of protein Y fluorescent you can determine that X and Y interact

Question 41

Each ligand receptor pair involves one specific ligand and one unique receptor, T or F

Answer 41

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

Question 42

Explain how activation of RTKs leads to signal transduction by the Ras pathway

Answer 42

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.

Question 43

Describe the structure of HSPGs

Answer 43

Consist of a proteoglycan core with glycosaminoglycan side chains