Receptor trafficking and protein interactions that regulate it 0.0 / 5 ? BiologyGeneticsUniversityNone Created by: molgCreated on: 17-05-18 16:50 AMPA GluA1 Drives insertion of proteins into membrane- LTP 1 of 25 AMPA GluA2/3 mainly involved in internalisation (LTD) 2 of 25 NMDA receptor activated by AMPA activity- triggers LTD/LTP 3 of 25 Receptor interacting proteins- LTD PICK1, NSF, AP2, ABP/GRIP- bound by accessory proteins 4 of 25 Receptor interacting proteins- LTP 4.1N, SAP-97, Myo V 5 of 25 Immunofluoresence GFP transfection- see outline of cell, then stained with antibody shows AMPA as spots 6 of 25 Antibody-feeding assays measure concentration of receptors on the membrane vs internalised receptors at a fixed time point 7 of 25 pHluorin-tagged receptor subunits monitor endocytosis/excytosis in real time- using pH-sensitive GFP- high fluorescence at neutral pH- loss at acidic (in lumen) 8 of 25 SEP = super ecliptic pHluorin 9 of 25 punctate regions at the surface membrane- higher receptor density- less internalisation 10 of 25 diffuse regions away from the membrane- lower receptor density- greater internalisation 11 of 25 GRIP stabilises proteins at the membrane and intracellular locations 12 of 25 PICK-1 binds to promote endocytosis (binds to same region as GRIP) 13 of 25 NSF binds to GluA2- controls interaction with PICK-1 14 of 25 Co-immunoprecipitation (CO-IP) show that 2 proteins interact in native tissues (eg PICK-1 and GluA2) 15 of 25 Co-localisation show that 2 proteins are in the same place in cells 16 of 25 SDS-PAGE proteins separated by gel according to molecular weight- acts as mesh filter 17 of 25 Sodium dodecyl sulphate (SDS) ionic detergent- coats protein in negative charge 18 of 25 Western blotting transfer proteins to nicrocellulose membrane- by electric current 19 of 25 Chemiluminescence enzyme-tagged-antibody (eg for GluA2) 20 of 25 immunofuorescence colocalisation 2 proteins are tagged with different coloured secondaries- proteins overlap produce different colour 21 of 25 analyse function of an interacting protein study trafficking or localisation of receptor by imaging/electrophysiology under various conditions; overexpressioon or "knock-down" by RNAi 22 of 25 overexpression engineered DNA into cells- virus, lipfection, Biolistics (gene-gun- gold bullets) 23 of 25 over expression of PICK-1 and GFP loss of surface GluA2 in the presence of extra PICK-1/ no change in total GluA2 24 of 25 "knock-down" of interacting protein expression RNA interference- siRNA/shRNA 25 of 25
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