16th Nov - Signalling Microdomains II Flashcards
Describe a signalling microdomain
Signalling complexes that form around activated receptors/
What are the components of the signalling complex that forms on an activated PDGFR
Src
PI3K
Grb 2 which recruits SOS through an SH3 domain
PLC gamma
What is an SH2 domain?
A protein module that helps create complexes which bind to specific phosphotyrosine motifs
Outline the structure of an SH2 domain
Has 2 binding site: one variable for c-term residues, and one conserved site for phosphotyrosines
Contains a central anti-parallel beta sheet surrounded by 2 alpha helices
What is the domain structure of the kinase src?
SH3 - SH2 - Kinase
What is the domain structure of the adaptor GRB2?
SH3 - SH2 - SH3
What is the domain structure of PLC gamma?
PH - PLC - SH2 - SH2 - SH3 -PLC
What does a Src-homology domain 2 bind to?
Short peptide motifs containing phopho-Y followe by 3-6aa C-term to pTyr
What does an Src-homology 3 domain bind to?
proline rich peptide sequences (P-X-X-P)
What does the pleckstrin homology bind to?
Phosphoinositides such as PIP2/PIP3
What does the PTB domain bind to?
The pTyr binding domain binds to motifs containing pY and 3-8aa n-terminal to pY
What does the PDZ domain bind to?
Binds to the last 4-5 C-terminal residues in the target protein, typically ion channels and receptors
Is NMDA-R a pre-formed signalling complex?
Yes
What are the advantages of having a stable pre-formed signalling complex e.g. NMDA-R?
It’s fast, efficient and specific
What is the main physiological function of having a pre-formed signalling complex on NMDA-R?
Fast synaptic transmission in the brain
What are the components of the NMDA-R pre-formed signalling complex?
NMDA-R is bound to Yotiao which binds PP1 and PKA
How did West (1999) attempt to elucidate Yotiao’s function?
They measured the current in the absence of yotiao –> no change in relative current upon application of glutamate
They measured the current with yotiao upon the application of glutamate –> Potentiation of current probably due to PKA
Used Ht31 to uncouple PKA from yotiao –> a dramatic decrease in peak current
–> Yotaio acts as a scaffold protein and phosphoryltion is not random
What did Welch show in 2010?
That Yotiao is an AKAP
What are AKAPs?
A-kinase anchor proteins which have the common function of binding to the regulatory subunit of protein kinase and localising it (And other proteins) to a form a signalling microdomain.
How many AKAPs are encoded in the mammalian genome?
75
Give an example of disease caused by mutation in a protein interaction domain leading to loss of protein protein interaction
Hypercholesteremia
Noonan Syndrome
Outline the mechanistic action of the immunosuppressant FK506
It inactivates pp2b (calcineurin) by creating an aberrant complex between calciuneurin and FKB12, causing calcineurin inactivation leading to interruption of signalling from the immune systems T-cell receptor
Outline the mechanistic action of rapamycin
Brings together FKBP12 and FRAP preventing FRAP from turning on the ribosomal kinase responsible for protein synthesis therefore inhibiting proliferation of T-lymphocytes
When was the SH2 domain identified?
1986
How is Heregulin regulated in human airway epithelial cells?
It is localise to the apical surface so that it is segregated from the ErbB2 receptor until polarity is disturbed
What is the approximate Kd of optimal SH2 binding?
about 50-500nM
How was Yotiao discoverd?
Through a Y2H screen for proteins that associated with NMDAR
How do only 20 AKAP genes produce 75 AKAPs in mammals?
Through alternative splicing
What is the function of AKAP 79/150?
Directs PKA to the beta adrenergic receptor
Co-ordinates PKC mediated phosphorylation –> angiotesin II induced hypertension
Physically associates with AC5 favouring phosporylation of the enzyme to terminate cAMP synthesis –> rapid terimation of cAMP signalling
