Activator of G-protein Signaling 3 (AGS3) modulates CXCR2/GRK6 activation and functions: Role of the GPR domain.

Abstract

Activator of G-protein Signaling 3 (AGS3) is a receptor-independent activator of G-protein signaling which comprises seven tetratricopeptide (TPR) motifs and four GPR motifs joined by a linker sequence. Previous studies have demonstrated that the TPR domain of AGS3 is important for determining the positioning of AGS3 within the cell, whereas the GPR domain serves as a docking site for Gαi-GDP leading to AGS3-Gαi signaling. We have recently shown that GRK6 complexes with AGS3-Gαi to regulate CXCR2-mediated cellular functions including Ca2+ mobilization, mitogen-activated protein kinase (MAPK) activation, receptor desensitization and down-regulation. In the current study, mutants from the GPR and TPR domains were used to determine which motif(s) of AGS3 interact with GRK6 to modulate CXCR2-mediated inflammatory responses. Co-immunoprecipitation studies in transiently transfected HEK 293 cells, and RBL-2H3 cells stably expressing wild-type and mutants AGS3 demonstrated that interleukin 8 (IL-8/CXCL8) activation increases GRK6 interaction with the GPR domain not the TPR. Interestingly, the TPR motif displayed a robust agonist-independent association with GRK6 which was prevented by CXCL8 activation of the receptor. Overexpression of AGS3 and the GPR motif, not the TPR, inhibited CXCR2 mediated intracellular Ca2+ mobilization and MAPK activation. Taken together, these results indicate that GRK6 interacts with the GPR domain of AGS3 to modulate CXCR2/Gai interaction and signal transduction.