Desensitization of GABAB receptor signaling by formation of protein complexes of GABAB2 subunit with GRK4 or GRK5

Abstract

We investigated the role of G protein coupled-receptor kinases (GRKs) in the desensitization of GABAB receptor-mediated signaling using Xenopus oocytes and baby hamster kidney (BHK) cells. Baclofen elicited inward K+ currents in oocytes coexpressing heterodimeric GABAB receptor, GABAB1a subunit (GB1aR) and GABAB2 subunit (GB2R), together with G protein-activated inwardly rectifying K+ channels (GIRKs), in a concentration-dependent manner. Repetitive application of baclofen to oocytes coexpressing GABABR and GIRKs did not change peak K+ 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 GABAB receptor-mediated desensitization processes. In BHK cells coexpressing GRK4 fused to Venus (brighter variant of yellow fluorescent protein, GRK4-Venus) with GB1aR and GB2R, GRK4-Venus was expressed in the cytosol but was translocated to the plasma membranes by GABABR activation. In BHK cells coexpressing GRK4 fused to Cerulean (brighter variant of cyan fluorescent protein, GRK4-Cerulean) with GB1aR and GB 2R-Venus, fluorescence resonance energy transfer (FRET) analysis demonstrated that GRK4-Cerulean formed a protein complex with GB 2R-Venus. Immunoprecipitation and Western blot analysis confirmed GB2R-GRK4 complex formation. GRK5 also formed a complex with GB 2R 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 GABAB receptor-mediated responses by forming protein complexes with GB2R subunit of GABABR at the plasma membranes. © 2006 Wiley-Liss, Inc.