Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Language
Publication year range
1.
Methods Cell Biol ; 132: 319-37, 2016.
Article in English | MEDLINE | ID: mdl-26928550

ABSTRACT

The use of biosensors either individually or as part of panels has now become a common technique to capturing signaling events in living cells. Such biosensors have become particularly important for probing biased signaling and allostery in G protein-coupled receptor drug screening efforts. However, assumptions about the portability of such biosensors between cell types may lead to misinterpretation of drug effects on specific signaling pathways in a given cellular context. Further, the output of a particular biosensor may be different depending on where it is localized in a cell. Here, we discuss strategies to mitigate these concerns which should feed into future biosensor design and usage.


Subject(s)
Biosensing Techniques , Signal Transduction , Cell Nucleus/enzymology , Enzyme Activation , Epidermal Growth Factor/pharmacology , Extracellular Signal-Regulated MAP Kinases/metabolism , Fluorescence Resonance Energy Transfer , HEK293 Cells , HeLa Cells , Humans , MAP Kinase Signaling System , Receptors, G-Protein-Coupled/metabolism , Tetradecanoylphorbol Acetate/pharmacology
2.
FASEB J ; 28(10): 4509-23, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25053617

ABSTRACT

G-protein-coupled receptors have been shown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo- and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo- and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of Gαi1, Gα13, Gαz, and ßarrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents Gi activation, also affects the CCR2-promoted engagement of Gαi1 and ßarrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo- and heteromultimeric CXCR4 and CCR2 can form functional signaling complexes that have unique properties.


Subject(s)
Arrestins/metabolism , Chemokine CCL2/metabolism , GTP-Binding Protein alpha Subunits/metabolism , Receptors, CCR2/metabolism , Receptors, CXCR4/metabolism , Signal Transduction , HEK293 Cells , Humans , Protein Binding , Protein Multimerization , Receptors, CXCR4/genetics , beta-Arrestins
SELECTION OF CITATIONS
SEARCH DETAIL
...