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Desensitization of GABA B receptor signaling by formation of protein complexes of GABA B2 subunit with GRK4 or GRK5
Author(s) -
Kanaide Masato,
Uezono Yasuhito,
Matsumoto Masanori,
Hojo Minoru,
Ando Yuko,
Sudo Yuka,
Sumikawa Koji,
Taniyama Kohtaro
Publication year - 2007
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.20863
Subject(s) - g protein coupled receptor kinase , biology , receptor , protein subunit , g protein coupled receptor , homologous desensitization , immunoprecipitation , microbiology and biotechnology , desensitization (medicine) , biophysics , chemistry , biochemistry , gene
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. J. Cell. Physiol. 210: 237–245, 2007. © 2006 Wiley‐Liss, Inc.