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This Article Full Text Full Text (PDF) All Versions of this Article: 580/1/51    most recent jphysiol.2006.123216v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fowler, C. E. Articles by Slesinger, P. A. Search for Related Content PubMed PubMed Citation Articles by Fowler, C. E. Articles by Slesinger, P. A. Related Collections Cellular

¹ Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA
² Departments of Neurosciences and Biology, University of California, La Jolla, CA, USA

Many neurotransmitters and hormones signal by stimulating G protein-coupled neurotransmitter receptors (GPCRs), which activate G proteins and their downstream effectors. Whether these signalling proteins diffuse freely within the plasma membrane is not well understood. Recent studies have suggested that direct protein–protein interactions exist between GPCRs, G proteins and G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels. Here, we used fluorescence resonance energy transfer (FRET) combined with total internal reflection fluorescence microscopy to investigate whether proteins within this signalling pathway move within 100 Å of each other in the plasma membrane of living cells. GABA_B R1 and R2 receptors, Kir3 channels, Go subunits and regulators of G protein signalling (RGS4) proteins were each fused to cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) and first assessed for functional expression in HEK293 cells. The presence of the fluorophore did not significantly alter the signalling properties of these proteins. Possible FRET was then investigated for different protein pair combinations. As a positive control, FRET was measured between tagged GABA_B R1 and R2 subunits (12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABA_B R1 or R2 subunits (13% FRET), and between Go and GABA_B R1 or R2 subunits (10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABA_B R1 or R2 subunits (10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Go. These data are discussed in terms of a model in which GABA_B receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.

(Received 19 October 2006; accepted after revision 14 December 2006; first published online 21 December 2006)
Corresponding author P. A. Slesinger: The Salk Institute, 10010 North Torrey Pines Rd, La Jolla, CA 92037, USA.  Email: slesinger{at}salk.edu

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