Desensitization of GABA(B) receptor signaling by formation of protein complexes of GABA(B2) subunit with GRK4 or GRK5.
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(+) 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(+) channels (GIRKs), in a concentration-dependent manner. Repetitive application of baclofen to oocytes coexpressing GABA(B)R 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 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.
Kanaide
Masato
M
Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
Uezono
Yasuhito
Y
Matsumoto
Masanori
M
Hojo
Minoru
M
Ando
Yuko
Y
Sudo
Yuka
Y
Sumikawa
Koji
K
Taniyama
Kohtaro
K
D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002477:Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000187:drug effects / Q000378:metabolism; D054771:G-Protein-Coupled Receptor Kinase 4; D054774:G-Protein-Coupled Receptor Kinase 5; D018755:GABA Agonists / Q000494:pharmacology; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects; D008845:Microinjections; D009842:Oligopeptides; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D018080:Receptors, GABA-B / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014981:Xenopus
D001426:Bacterial Proteins; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D018755:GABA Agonists; D008164:Luminescent Proteins; D009842:Oligopeptides; D010455:Peptides; D021122:Protein Subunits; D018080:Receptors, GABA-B; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; C085706:FLAG peptide; D017346:Protein Serine-Threonine Kinases; D054771:G-Protein-Coupled Receptor Kinase 4; D054774:G-Protein-Coupled Receptor Kinase 5; D001418:Baclofen