Homodimerization Of G Protein-coupled Receptor Kinase 6 And Its Role In Plasma Membrane Targeting

Objective: G protein-coupled receptor kinases( GRKS) specifically phosphorylate ligand-bound G protein-coupled receptors(GPCRS)and rapidly shut-off or desensitize the agonist-triggered signal transduction, which plays an important roles in regulation of the pathophysiological activities of GPCRs. GRK6 is a member of GRK4 subfamily of GRKs and is known to have diverse cellular functions ranging from metabolism to growth and neuron degeneration. The crystal structure of GRK6 shows that its N terminal G protein signal regulator(RGS) domain forms an extensive dimeric interface using several hydrophobic residues. Mutation of those residues could decrease the kinase activity of GRK6. Therefore, homodimerization could be a critial mechanism of GRK6 in regulating GPCR signaling. To date, the evidence of GRK6 homodimerization in cells has not been evidenced yet. On the other hand, plasma membrane localization is a prerequisite for GRK6 to regulate GPCR signal pathway. However, the influence of homodimerization on plasma membrane targeting of GRK6 is totally unclear. This study is aimed to reveal the phenomenon and mechanism of homodimerization of GRK6 in cells, as well as its influence on plasma membrane targeting. Methods: Firstly, we used bimolecular fluorescence complementation(Bi FC)and Bi FC competition(Bi FC-C)assays, combined with an acceptor photo-bleaching fluorescence resonance energy transfer(FRET) assay to test the self-association of GRK6. For this end, human full-length GRK6 c DNA was respectively cloned into Bi FC vectors p Bi FC-VN173/ p Bi FC-VC155 pair and the EGFP/m Cherry pair ofFRET. The Bi FC signal and the FRET efficiency were analyzed by flowcytometry and confocal laser scanning microscopy, respectively, in HEK293 cells. Secondly, we mutated the Met165, Tyr166 and Phe527 residues of GRK6(GRK6EED) and compared the Bi FC signal as well as the FRET efficiency of the mutants with those of wild type GRK6. At last, the membrane localization of the dimeric interface mutants was tested by fluorescence microscopy. Results: In the cells of coexpression of GRK6-VN173 + GRK6-VC155, strong positive Bi FC signals were detected; both the number of positive cells and the intensity of Bi FC signals could be significantly inhibited by competitor GRK6-m Cherry. The FRET signals were detected when GRK6-GFP and GRK6-m Cherry proteins coexpressed, while in group of GRK6 EED, both the cellular Bi FC signals and the FRET efficiency were significantly lower than those in wild type GRK6 group; the GRK6 EED lost the ability of plasma membrane targeting and mainly distributed in cytoplasm. Conclusion: Our results show that GRK6 can form homodimers in cells. The Met165, Tyr166 and Phe527 may be the critical residues for the homodimerization, which plays an important role in plasma membrane localization of GRK6. Our research provides direct evidences of homodimerization of GRK6 in cells, which suggests a new mechanism underlying the subcellular distribution of GRK6.