16th Nov - Signalling Microdomains II

Question 1

Describe a signalling microdomain

Answer 1

Signalling complexes that form around activated receptors/

Question 2

What are the components of the signalling complex that forms on an activated PDGFR

Answer 2

Src
PI3K
Grb 2 which recruits SOS through an SH3 domain
PLC gamma

Question 3

What is an SH2 domain?

Answer 3

A protein module that helps create complexes which bind to specific phosphotyrosine motifs

Question 4

Outline the structure of an SH2 domain

Answer 4

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

Question 5

What is the domain structure of the kinase src?

Answer 5

SH3 - SH2 - Kinase

Question 6

What is the domain structure of the adaptor GRB2?

Answer 6

SH3 - SH2 - SH3

Question 7

What is the domain structure of PLC gamma?

Answer 7

PH - PLC - SH2 - SH2 - SH3 -PLC

Question 8

What does a Src-homology domain 2 bind to?

Answer 8

Short peptide motifs containing phopho-Y followe by 3-6aa C-term to pTyr

Question 9

What does an Src-homology 3 domain bind to?

Answer 9

proline rich peptide sequences (P-X-X-P)

Question 10

What does the pleckstrin homology bind to?

Answer 10

Phosphoinositides such as PIP2/PIP3

Question 11

What does the PTB domain bind to?

Answer 11

The pTyr binding domain binds to motifs containing pY and 3-8aa n-terminal to pY

Question 12

What does the PDZ domain bind to?

Answer 12

Binds to the last 4-5 C-terminal residues in the target protein, typically ion channels and receptors

Question 13

Is NMDA-R a pre-formed signalling complex?

Answer 13

Yes

Question 14

What are the advantages of having a stable pre-formed signalling complex e.g. NMDA-R?

Answer 14

It’s fast, efficient and specific

Question 15

What is the main physiological function of having a pre-formed signalling complex on NMDA-R?

Answer 15

Fast synaptic transmission in the brain

Question 16

What are the components of the NMDA-R pre-formed signalling complex?

Answer 16

NMDA-R is bound to Yotiao which binds PP1 and PKA

Question 17

How did West (1999) attempt to elucidate Yotiao’s function?

Answer 17

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

Question 18

What did Welch show in 2010?

Answer 18

That Yotiao is an AKAP

Question 19

What are AKAPs?

Answer 19

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.

Question 20

How many AKAPs are encoded in the mammalian genome?

Answer 20

75

Question 21

Give an example of disease caused by mutation in a protein interaction domain leading to loss of protein protein interaction

Answer 21

Hypercholesteremia

Noonan Syndrome

Question 22

Outline the mechanistic action of the immunosuppressant FK506

Answer 22

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

Question 23

Outline the mechanistic action of rapamycin

Answer 23

Brings together FKBP12 and FRAP preventing FRAP from turning on the ribosomal kinase responsible for protein synthesis therefore inhibiting proliferation of T-lymphocytes

Question 24

When was the SH2 domain identified?

Answer 24

1986

Question 25

How is Heregulin regulated in human airway epithelial cells?

Answer 25

It is localise to the apical surface so that it is segregated from the ErbB2 receptor until polarity is disturbed

Question 26

What is the approximate Kd of optimal SH2 binding?

Answer 26

about 50-500nM

Question 27

How was Yotiao discoverd?

Answer 27

Through a Y2H screen for proteins that associated with NMDAR

Question 28

How do only 20 AKAP genes produce 75 AKAPs in mammals?

Answer 28

Through alternative splicing

Question 29

What is the function of AKAP 79/150?

Answer 29

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