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In JoVE (1)
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Articles by Jason S. Kerr in JoVE
JoVE General
Avidez baseado Triagem Interação Extracelular (AVEXIS) para a detecção escalável de baixa afinidade Extracelular receptor-ligante Interações
Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute
AVEXIS é uma alta taxa de ensaio interação proteína desenvolvida para a tela de forma sistemática para novas extracelular do receptor-ligante pares envolvidos em processos de reconhecimento celular. Ele é projetado especificamente para detectar interações protéicas transitórios que são difíceis de identificar com outras abordagens de alto rendimento.
Other articles by Jason S. Kerr on PubMed
CCR5 Internalisation and Signalling Have Different Dependence on Membrane Lipid Raft Integrity
The chemokine receptor, CCR5, acts as a co-receptor for human immunodeficiency virus entry into cells. CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains termed lipid rafts or caveolae. Cholesterol is essential for CCL4 binding to CCR5 and for keeping the conformational integrity of the receptor. Filipin treatment leads to loss of caveolin-1 from the membrane and therefore to a collapse of the caveolae. We have found here that sequestration of membrane cholesterol with filipin did not affect receptor signalling, however a loss of ligand-induced internalisation of CCR5 was observed. Cholesterol extraction with methyl-beta-cyclodextrin (MCD) reduced signalling through CCR5 as measured by release of intracellular Ca(2+) and completely abolished the inhibition of forskolin-stimulated cAMP accumulation with no effect on internalisation. Pertussis toxin (PTX) treatment inhibited the intracellular release of calcium that is transduced via Galphai G-proteins. Depletion of cholesterol destroyed microdomains in the membrane and switched CCR5/G-protein coupling to a PTX-independent G-protein. We conclude that cholesterol in the membrane is essential for CCR5 signalling via the Galphai G-protein subunit, and that integrity of lipid rafts is not essential for effective CCR5 internalisation however it is crucial for proper CCR5 signal transduction via Galphai G-proteins.
