Protein - Protein Interactions

Question 1

What are the two types of signalling complexes?

Answer 1

Ones which are formed following receptor activation e.g. RTKs such as PDGF
Ones which are a preformed stable complex e.g. NMDAR yotiao complex

Question 2

What does SH2 bind to?

Answer 2

Mainly phosphorylated tyrosine

Question 3

What occurs on ligand binding to a RTK?

Answer 3

Dimerisation
TK domains brought close together to cause cross auto phosphorylation of tyrosine residues
Allows interaction with signalling molecules
Recruitment of signalling molecules to phospho tyrosine residues

Question 4

SH2 domains can bind to phosphorylated tyrosine, what else is important?

Answer 4

SH2 domain also recognises down stream amino advice sequences too which allows a level of specificity i.e. SH2 may recognise YIYV

Question 5

What proteins contain an SH2 domain?

Answer 5

Src
Grb2
PI3K
PLC-Y1

Question 6

What is Grb2?

Answer 6

Acts as an adaptor protein which has an SH2 and SH3 domain so RTK activation can activate SH3 mediated signals too

Question 7

Generally what are the effects of RTK activation?

Answer 7

Cell growth
Cell survival
Cell migration

Question 8

Where are the N and C terminus of the SH2 domain located?

Answer 8

Juxtaposed to eachother

Question 9

How many amino acids make up the SH2 domain?

Answer 9

About 100

Question 10

How many proteins contain a SH2 domain

Answer 10

About 120

Question 11

What is special about the SH2 domain amought all protein which contain one?

Answer 11

They all contain a conserved area which binds to phosphorylated tyrosine

Question 12

Part of the SH2 domain recognises 3-6 amino acid residues downstream of the phosphotyrosine…what is important about this area?

Answer 12

It allows a level of specificity (binding requires recognition of a sequence not just a phosphorylated tyrosine)
This is a variable region in the SH2 domain

Question 13

What other domains allow proteion protein interactions?

Answer 13

SH3 (PXXP)
Pleckstrin homology domains (PIP2 and PIP3)
PDZ domains (XT/SXV)

Question 14

What do preformed signalling complexes allow for?

Answer 14

Signalling which is specific, quick and efficient

Question 15

What family of proteins does yotaio belong to?

Answer 15

A-Kinase anchoring proteins (AKAP)

Question 16

How many AKAPs are there ?

Answer 16

About 20 with 75 alternative splice variants

Question 17

What AKAP is important for B2AR+AC+Cav1.2 coupling to PKA?

Answer 17

AKAP 79

Question 18

What does yotaio bind to?

Answer 18

NMDAR
PKA (increases NMDAR activity)
PP1 (decreases NMDAR activity)

Activation of PP1 and PKA depends on intracellular signals therefore intracellular signals can modulate channel activity

Question 19

What did Westphal et al 1999 do?

Answer 19

Applied glutamate to cell whilst makin electrophysological measurements.
Cells without yotaio did not show an increase in glutamate mediated current when PKA was activated (addition of cAMP)
Cells with yotiao did show an increase in glutamate mediate dcurrent on action of cAMP

Question 20

What attenuated cAMP mediated increase in NMDAR current? Why?

Answer 20

Ht31 as this uncoupled PKA from yotiao.

Indicates that increase in current mediated by PKA being localised to the NMDAR

Question 21

How can protein - protein interactions become pathogenic?

Answer 21

Can causes hypercholesterolaemia through mutant human ARH protein in the PTB domain prevents LDL receptors associating with endocytic machinery
Formation of fusion proteins such as BCR-ABL can promote protein protein interactions
E.g. All autophosphylates tyrosine 117 which allows SH2 + grb2 interaction and signalling

Question 22

What are the potential therapeutic targets for protein protein interactions? Give an example

Answer 22

Possible to form interactions through addition of exogenous substance which may sequester protein signalling
E.g. Immunosuppressive tacrolimus allows a protein protein interaction between Calcineurin and FKBP12 which decreases calcineurin mediated increase in IL-2 = decrease T cell responce