Receptor trafficking and protein interactions that regulate it

  • Created by: molg
  • Created on: 17-05-18 16:50
AMPA GluA1
Drives insertion of proteins into membrane- LTP
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AMPA GluA2/3
mainly involved in internalisation (LTD)
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NMDA receptor
activated by AMPA activity- triggers LTD/LTP
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Receptor interacting proteins- LTD
PICK1, NSF, AP2, ABP/GRIP- bound by accessory proteins
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Receptor interacting proteins- LTP
4.1N, SAP-97, Myo V
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Immunofluoresence
GFP transfection- see outline of cell, then stained with antibody shows AMPA as spots
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Antibody-feeding assays
measure concentration of receptors on the membrane vs internalised receptors at a fixed time point
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pHluorin-tagged receptor subunits
monitor endocytosis/excytosis in real time- using pH-sensitive GFP- high fluorescence at neutral pH- loss at acidic (in lumen)
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SEP
= super ecliptic pHluorin
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punctate regions
at the surface membrane- higher receptor density- less internalisation
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diffuse regions
away from the membrane- lower receptor density- greater internalisation
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GRIP
stabilises proteins at the membrane and intracellular locations
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PICK-1
binds to promote endocytosis (binds to same region as GRIP)
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NSF
binds to GluA2- controls interaction with PICK-1
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Co-immunoprecipitation (CO-IP)
show that 2 proteins interact in native tissues (eg PICK-1 and GluA2)
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Co-localisation
show that 2 proteins are in the same place in cells
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SDS-PAGE
proteins separated by gel according to molecular weight- acts as mesh filter
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Sodium dodecyl sulphate (SDS)
ionic detergent- coats protein in negative charge
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Western blotting
transfer proteins to nicrocellulose membrane- by electric current
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Chemiluminescence
enzyme-tagged-antibody (eg for GluA2)
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immunofuorescence colocalisation
2 proteins are tagged with different coloured secondaries- proteins overlap produce different colour
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analyse function of an interacting protein
study trafficking or localisation of receptor by imaging/electrophysiology under various conditions; overexpressioon or "knock-down" by RNAi
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overexpression
engineered DNA into cells- virus, lipfection, Biolistics (gene-gun- gold bullets)
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over expression of PICK-1 and GFP
loss of surface GluA2 in the presence of extra PICK-1/ no change in total GluA2
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"knock-down" of interacting protein expression
RNA interference- siRNA/shRNA
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Other cards in this set

Card 2

Front

AMPA GluA2/3

Back

mainly involved in internalisation (LTD)

Card 3

Front

NMDA receptor

Back

Preview of the front of card 3

Card 4

Front

Receptor interacting proteins- LTD

Back

Preview of the front of card 4

Card 5

Front

Receptor interacting proteins- LTP

Back

Preview of the front of card 5
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