Evidence for association of GABAB receptors with Kir3 channels and regulators of G protein signalling (RGS4) proteins

Authors


Corresponding author P. A. Slesinger: The Salk Institute, 10010 North Torrey Pines Rd, La Jolla, CA 92037, USA.  Email: slesinger@salk.edu

Abstract

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. GABAB R1 and R2 receptors, Kir3 channels, Gαo 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 GABAB R1 and R2 subunits (∼12% FRET), which are known to form heterodimers. We measured significant FRET between tagged RGS4 and GABAB R1 or R2 subunits (∼13% FRET), and between Gαo and GABAB R1 or R2 subunits (∼10% FRET). Surprisingly, FRET also occurred between tagged Kir3.2a/Kir3.4 channels and GABAB R1 or R2 subunits (∼10% FRET). FRET was not detected between Kir3.2a and RGS4 nor between Kir3.2a and Gαo. These data are discussed in terms of a model in which GABAB receptors, G proteins, RGS4 proteins and Kir3 channels are closely associated in a signalling complex.

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