title: G Protein-coupled receptor kinase-6 interacts with activator of G protein signaling-3 to regulate CXCR2-mediated cellular functions.
abstract: The IL-8 (CXCL8) receptors CXCR1 and CXCR2 couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. We recently showed that CXCR1 couples predominantly to the G protein-coupled receptor kinase (GRK)2, whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to G protein-coupled receptors, GRKs displayed a more diverse protein/protein interaction in cells. In this study, we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. Our data demonstrated that, upon CXCR2 activation, GRK6 interacts with activator of G protein signaling (AGS)3 and Gαi2 to form a GRK6/AGS3/Gαi2 complex. This complex is time dependent and peaked at 2-3 min postactivation. GTPγS pretreatment blocked GRK6/AGS3/Gαi2 formation, suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent, but GRK6-independent, fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca(2+) mobilization, phosphoinositide hydrolysis, and chemotaxis. In contrast, short hairpin RNA inhibition of AGS3 enhanced CXCL8-induced Ca(2+) mobilization, receptor resistance to desensitization, and recycling to the cell surface, with no effect on receptor internalization. Interestingly, RBL-CXCR2-AGS3(-/-) cells displayed a significant increase in CXCR2 expression on the cell surface but decreased ERK1/2 and P38 MAPK activation. Taken together, these results indicate that GRK6 complexes with AGS3-Gαi2 to regulate CXCR2-mediated leukocyte functions at different levels, including downstream effector activation, receptor trafficking, and expression at the cell membrane.
authors: Department of Biology, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, Durham, NC 27707.
authors: Vandana
authors: V
authors: Singh
authors: Sandeep K
authors: SK
authors: Raghuwanshi
authors: Nikia
authors: N
authors: Smith
authors: Elizabeth J
authors: EJ
authors: Rivers
authors: Ricardo M
authors: RM
authors: Richardson
mesh_terms: D000818:Animals; D002118:Calcium / Q000276:immunology; D020013:Calcium Signaling / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology; D054768:G-Protein-Coupled Receptor Kinases / Q000235:genetics / Q000276:immunology*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000276:immunology; D005786:Gene Expression Regulation / Q000502:physiology; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000276:immunology*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D046912:Multiprotein Complexes / Q000235:genetics / Q000276:immunology*; D010766:Phosphorylation / Q000235:genetics / Q000276:immunology; D021381:Protein Transport / Q000502:physiology; D023063:Receptors, Interleukin-8B / Q000235:genetics / Q000276:immunology*
chemical_list: C525615:GPSM1 protein, human; D044385:GTP-Binding Protein alpha Subunits; D020730:Guanine Nucleotide Dissociation Inhibitors; D046912:Multiprotein Complexes; D023063:Receptors, Interleukin-8B; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; D002118:Calcium