Receptor Mechanism III Flashcards
Describe the structural features of the different kinase or kinase linked receptors
1) Tyrosine receptors kinase (activates enzyme activity)
o Substrate for kinase are specific tyrosine residues which are phosphorylated
o EGF (epidermal growth factor)
o PDGF (platelet derived growth factor)
o Insulin (slight different)
2) JAK/STAT
o Growth hormone
o Interferon
3) Serine Threonine receptor kinase
o TGFβ
Describe the events that take place following the binding of the agonist to the receptor
• Two receptor molecules, and two identical growth factors (ligands) that bind to these receptors
• Binding of the ligand to receptor causes conformational change, which allow two identical molecules come together to allow dimerization.
o Dimerization – simplest form of interaction that takes place
o This therefore brings the two receptors into very close contact with each other
o Receptors normally move within lipid bilayer, but activation (where binding of the ligands to receptors occurs), facilitates this dimerization
o Dimerization brings together elements within two receptors molecules and allows them to modify its partner.
• Modification that takes place is phosphorylation.
o Since the receptor is phosphorylating itself, it is called auto-phosphorylation
o Purpose of phosphorylation signals to other proteins that that receptor is activated
o Phosphorylation itself causes conformational changes which allows receptor to interact with other proteins
• Chain of events:
o Activation of receptor leads to conformational change
o Phosphorylation leads to binding of another protein
o This can possibly lead to phosphorylation of that protein but definitely a conformational change
o This can cause the overall activation of another molecule
• Phosphorylation provides conformational change which provides a docking site for other proteins. These proteins would otherwise not be able to bind can bind, and so are activated
• Type of activation depends on type of protein which has bound
Give examples of growth factors and cytokines that act via the different receptor types.
• Predominantly single transmembrane domain receptors – not multiple like 7 transmembrane
• Activation leads to activation of a receptor kinase
o directly to enzyme activity within receptor molecule itself or very closely associated protein
• Activation leads to the activation of multiple signalling pathways
o unlike GPCR which generates a single intracellular message, these receptors can often activate multiple pathways
o Which pathway activated, depends on cellular environment receptor is in.
Discuss the common consequences of mutations in the receptors
• Many of the proteins in these pathways are altered in disease state
o Mutations in Ras leads to over activation
o Mutation in receptor – where receptor doesn’t need agonist to bind for receptor to be active
o Loss of GTPase activity
o Loss of Phosphatase activity – mutations in proteins such as Pten – prolonged activation of cascade activated by receptor
• Many of these proteins are potential targets for therapy
o If EGF binds to receptor it will stimulate this cascade
o One of the common targets for treatments of breast cancer is EGF receptor
o Herceptin is an antibody therapy that will interact and interfere with activation of this receptor and receptor cascade
Enzyme linked receptors are
• Involved in regulation of o Cell growth o Cell Division o Differentiation o Cell survival o Cell migration o Regulating Metabolic pathways
- Important because inappropriate activation is associated with diseases such as cancer
- Important targets for therapy
Tyrosine kinases activity
- Provides a site which is common to a lot of different molecules
- Basic structural homology in many proteins
- Within receptor molecule are tyrosine residues
- Tyrosine kinase phosphorylates tyrosine residues
- Tyrosine phosphorylation is extremely specific – not all phosphorylated
- Kinase will recognise particular orientation of tyrosine and identify its shape to phosphorylate
- This causes conformational change which will present this docking site.
- This docking site will then allow interaction with a second protein
- This second protein has a complementary conformation which will bind to the activated tyrosine molecule.
- This part of the second protein is called SH2 domain
- Many SH2 domains from different proteins – all have a basic common structure, even though they may have some specific differences.
- These proteins with SH2 domains will bind to and be activated by the activated receptor.
- It will be specific proteins binding to specific regions
- If molecularly manipulate protein, and got rid of a tyrosine, a particular protein will not be able to bind.
Ras protein
- There is a small GTP binding protein called “Ras”
- Activation of a receptor will form regions which can then be recognised by another protein, which binds to it, and undergoes a conformational change
- Binding will be through these SH2 domains.
- Then another conformational change allows another protein, which in this instance, will facilitate activation of the Ras protein
- Interaction of the GRB-2 and Ras GEF proteins is by SH3 domains
- Activation of this protein like other G-proteins is by displacing GDP with GTP
- Activated Ras, will then initiate a number of different intracellular signals
- Activation of Ras GEF protein, facilitates the exchange - increases the activity
PI 3-kinase
• Most common – Activated PI 3-kinase protein
• PI 3-kinase phosphorylates membrane lipids (specifically PIP2)
• This produces PIP3, and can bind to and activate specific proteins
• Here, it activates two proteins
o PDK1 protein which phosphorylates and activates a second protein called PKB (Protein Kinase B).
o PKB dissociates and activates other molecules within site
Insulin receptor
• Acts on liver and muscle to reduce blood glucose
• Same receptor- different results
o Liver – glucose uptake is not insulin dependent. Deposition into glycogen and fatty acids is dependent on insulin
o Muscle- uptake can be stimulated by insulin
• Insulin receptor consists of 2 alpha and 2 beta subunits linked by disulphide bridges
• Binding of insulin to receptor will activate tyrosine kinase and you get auto-phosphorylation of 2 beta sub-unit
• Activation of receptor leads to activation of family of small protein substrates called Insulin Receptor Substrate (IRS)
• Binding of insulin causes conformational change, but only brings together the two beta subunits rather than bringing two receptor molecules together
• The IRS binds to two similar SH2 domain interactions
• IRS activates the PI 3-kinase
• Get activation of PDK1 which then activates PKB.
• In muscles, the PKB activation can lead to many events
o movement of receptors to the cell surface, allowing glucose uptake to increase
o Synthesis of glycogen
How are receptor kinases inactivated (cellular control)?
1) As activation of these receptors leads to phosphorylation, de-phosphorylation leads to inactivation
2) Phosphatases are activated as a result of receptor activation.
• Phosphatases remove these phosphates and disables interaction with other proteins
3) Signalling processes lead to signal termination
• Phosphatases which prevent or remove phosphorylation event in terms of activation of PI 3-kinase.
• Pten is a phosphatase, which inhibits activations of PKB
4) GTP binding proteins have associated with them ATPase activity
5) Receptor internalisation (most common)
• Removing receptor from the surface by breaking down or removing agonist/phosphate from receptor/ intracellular organelles and exporting them back to cell surface in inactive form
Small G proteins
- GEF (guanine nucleotide exchange factor) protein is activated and helps in activation of Ras
- Increases activity significantly
- Most cancers have a mutation in Ras protein in common. This mutation inhibits the GTPase activity – prevents inactivation of G-protein
Regulation of Ras activity
- Activation occurs by the exchange of GDP for GTP
- Assisted by exchange factor (GEF) which results in phosphorylation
- Activated Ras functions for a period of time
- Has activity that hydrolyses the GTP gradually
- Activation will recruit a GTPase activating protein which aids hydrolysis of GTP to GDP
- This association is relatively slow in molecular terms.
- This leads to removal of phosphate and inactivation of Ras.
- Then converted back to its inactive state wait for next activation
JAK-STAT signalling pathway activated by Growth hormone and some cytokines
- Single transmembrane domain protein receptor
- Unlike tyrosine kinase, this receptor doesn’t have any enzyme activity associated with it
- It has another protein which is closely associated, which has this tyrosine kinase activity called JAK
- JAK – Janus kinase (means two heads)
- JAK acts exact same way as an agonist (interferon or growth hormone binds). Get dimerization
- Association of JAK means it can also phosphorylate which is signified by no.3
- This leads to phosphorylation of receptor
- Which allows association of another protein called STAT
- Binding of STAT will lead to conformational change and phosphorylation of STAT
- This allows two molecules of STAT to dimerise
- The dimerised STAT then transported and move into nucleus where it binds to specific sites on DNA causing an increase in gene transcription.
Smad-dependant signalling pathway activated by TGF-β
- Two receptors, dimerization of two different receptor molecules
- TGFβ is present as a dimer and initially binds to receptor 2.
- Within the receptor is serine-threonine kinase activity (not tyrosine)
- Binding allows association of receptor 1 and receptor 2
- Receptor 1 undergoes phosphorylation in response to binding.
- This particular activation of this receptor leads to associating with molecules called Smad.
- Activation of Smad 2 and Smad 3 allow it to activate or interact with Smad 4
- As a heterodimer, Smad is translocated into the nucleus, where it regulates gene expression
cAMP vs inositolP s enzyme linked signal transduction pathways
Conformational change in G protein/ G protein/ and phosphorylation
Adenyl cyclase / phospholipase C / proteins with SH2 domains
GTPase phosphodiesterases / GTPase phosphorylation / phosphatase
