Abstract
Regulators of G-protein signaling (RGS) proteins act directly on Galpha subunits to increase the rate of GTP hydrolysis and to terminate signaling. However, the mechanisms involved in determining their specificities of action in cells remain unclear. Recent evidence has raised the possibility that RGS proteins may interact directly with G-protein-coupled receptors to modulate their activity. By using biochemical, fluorescent imaging, and functional approaches, we found that RGS2 binds directly and selectively to the third intracellular loop of the alpha1A-adrenergic receptor (AR) in vitro, and is recruited by the unstimulated alpha1A-AR to the plasma membrane in cells to inhibit receptor and Gq/11 signaling. This interaction was specific, because RGS2 did not interact with the highly homologous alpha1B- or alpha1D-ARs, and the closely related RGS16 did not interact with any alpha1-ARs. The N terminus of RGS2 was required for association with alpha1A-ARs and inhibition of signaling, and amino acids Lys219, Ser220, and Arg238 within the alpha1A-AR i3 loop were found to be essential for this interaction. These findings demonstrate that certain RGS proteins can directly interact with preferred G-protein-coupled receptors to modulate their signaling with a high degree of specificity.
Highlights
Signaling through G-protein-coupled receptors (GPCRs)1 must be tightly regulated in cells to maintain functional specificity
RGS2 Selectively Associates with the i3 Loop of the ␣1AAR—To determine whether selected B/R4 Regulators of G-protein signaling (RGS) proteins directly associate with ␣1-adrenergic receptor (AR), we examined the capacity of closely related RGS2 and RGS16 to associate with the i3 loops of all three subtypes (␣1A, ␣1B, and ␣1D) by using pull-down assays
RGS2 Co-localizes at the Plasma Membrane with ␣1A-ARs in HEK293 Cells—To determine whether RGS2 associates with ␣1A-ARs in a cellular context, we co-transfected GFP-tagged RGS proteins with HA-tagged ␣1-ARs into HEK293 cells, and we examined their cellular localization by using confocal microscopy
Summary
Constructs-GST-␣1-i3 Constructs—␣1A-i3, ␣1B-i3, and ␣1D-i3 loop constructs were originally cloned into the pET41b vector to encode a fusion protein with an N-terminal GST tag and a C-terminal His tag. In order to perform pull-down assays with purified His-tagged RGS proteins, fragments encoding the i3 loops of ␣1A- and ␣1B-ARs were subcloned further into the pGEX4T vector to eliminate the His tag. GFP-tagged constructs were grown on sterile coverslips, fixed for 30 min with 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and rinsed three times with phosphate-buffered saline (PBS) containing 0.5% normal horse serum (PBSϩ). Anti-HA antibody (1:1000 dilution) was added to coverslips overnight at 4 °C in blocking buffer, washed three times with PBSϩ, and incubated with rhodamine red-conjugated antirabbit IgG secondary antibody (1:500 dilution) for 1 h at room temperature in blocking buffer. Transfected HEK293 cells were prelabeled with myo-[3H]inositol for 24 h, and production of [3H]InsP was determined by modification of a protocol described previously [45]. Data were analyzed using nonlinear regression [26]
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