Desensitization of GABA_B receptor signaling by formation of protein complexes of GABA_B2 subunit with GRK4 or GRK5

Abstract

We investigated the role of G protein coupled-receptor kinases (GRKs) in the desensitization of GABA B receptor-mediated signaling using Xenopus oocytes and baby hamster kidney (BHK) cells. Baclofen elicited inward K þ currents in oocytes coexpressing heterodimeric GABA B receptor, GABA B1a subunit (GB 1a R) and GABA B2 subunit (GB 2 R), together with G protein-activated inwardly rectifying K þ channels (GIRKs), in a concentration-dependent manner. Repetitive application of baclofen to oocytes coexpressing GABA B R and GIRKs did not change peak K þ currents in the first and second responses, but the latter responses were significantly attenuated by coexpression of either GRK4 or GRK5 with attenuation efficacy of GRK4 > GRK5. Coexpression of other GRKs including GRK2, GRK3, and GRK6 had no effect on GABA B receptor-mediated desensitization processes. In BHK cells coexpressing GRK4 fused to Venus (brighter variant of yellow fluorescent protein, GRK4-Venus) with GB 1a R and GB 2 R, GRK4-Venus was expressed in the cytosol but was translocated to the plasma membranes by GABA B R activation. In BHK cells coexpressing GRK4 fused to Cerulean (brighter variant of cyan fluorescent protein, GRK4-Cerulean) with GB 1a R and GB 2 R-Venus, fluorescence resonance energy transfer (FRET) analysis demonstrated that GRK4-Cerulean formed a protein complex with GB 2 R-Venus. Immunoprecipitation and Western blot analysis confirmed GB 2 R-GRK4 complex formation. GRK5 also formed a complex with GB 2 R on the plasma membranes as determined by FRET and Western blotting but not GRK2, GRK3, and GRK6. Our results indicate that GRK4 and GRK5 desensitize GABA B receptor-mediated responses by forming protein complexes with GB 2 R subunit of GABA B R at the plasma membranes.

μ-Opioid Receptor Forms a Functional Heterodimer With Cannabinoid CB1 Receptor: Electrophysiological and FRET Assay Analysis

“…For transfection experiments, BHK cells were seeded at a density of 1 × 10 5 cells / 35-mm glass-bottomed culture dish (World Precision Instrument, Sarasota, FL, USA) for 24 h. For western blot assay, cells were seeded at a density of 1 × 10 6 cells / 35-mm dish. Transient transfection was then performed with Effectene transfection reagent (Qiagen, Tokyo) containing 0.2 μg each of cDNAs as described previously (11,15). Cells were used in confocal microscopy analysis, FRET analysis, and Western blotting assay 16 -24 h after transfection.…”

“…FRET was measured by imaging Cerulean before and after photo-bleaching Venus with the 100% intensity of 514-nm argon laser for 30 -60 s, a duration that efficiently bleached Venus with little effect on Cerulean (11,15). An increase of the donor fluorescence (Cerulean) was interpreted as evidence of FRET from Cerulean to Venus.…”

“…1). FRET efficiency was then calculated using the equation E = 1 − I DA / I D , where I DA is the peak of donor emission in the presence of the acceptor and I D is the peak in the presence of the sensitized acceptor, as described previously (11,15,17). Before and after this bleaching, Cerulean images were collected to assess changes in donor fluorescence.…”

“…BHK cells were transiently cotransfected with each of the FLAG-tagged rat μOR cDNA and human CB 1 R cDNA, alone or both as described previously (15). Briefly, 24 h after transfection, BHK cells were harvested, sonicated, and solubilized in the PROREP protein extraction buffer containing a protease inhibitor cocktail (iNtRON Biotechnology, Sungnam, Korea) for 1 h at 4°C.…”

Tolerance to continuous intrathecal baclofen infusion can be reversed by pulsatile bolus infusion

“…9 The hypothesis is that tolerance in ITB therapy is induced by a reduction of the total number of GABA B receptors, and/or by GABA B receptor desensitization. [10][11][12] The key question is how to treat tolerance in ITB therapy optimally. Altering the infusion mode from simple to complex continuous has shown not to be effective in the development of tolerance.…”