title: Selective recruitment of G protein-coupled receptor kinases (GRKs) controls signaling of the insulin-like growth factor 1 receptor.
abstract: β-Arrestins are multifunctional proteins that play central roles in G protein-coupled receptor (GPCR) trafficking and signaling. β-Arrestin1 is also recruited to the insulin-like growth factor-1 receptor (IGF-1R), a receptor tyrosine kinase (RTK), mediating receptor degradation and signaling. Because GPCR phosphorylation by GPCR-kinases (GRKs) governs interactions of the receptors with β-arrestins, we investigated the regulatory roles of the four widely expressed GRKs on IGF-1R signaling/degradation. By suppressing GRK expression with siRNA, we demonstrated that lowering GRK5/6 abolishes IGF1-mediated ERK and AKT activation, whereas GRK2 inhibition increases ERK activation and partially inhibits AKT signaling. Conversely, β-arrestin-mediated ERK signaling is enhanced by overexpression of GRK6 and diminished by GRK2. Similarly, we demonstrated opposing effects of GRK2 and -6 on IGF-1R degradation: GRK2 decreases whereas GRK6 enhances ligand-induced degradation. GRK2 and GRK6 coimmunoprecipitate with IGF-1R and increase IGF-1R serine phosphorylation, promoting β-arrestin1 association. Using immunoprecipitation, confocal microscopy, and FRET analysis, we demonstrated β-arrestin/IGF-1R association to be transient for GRK2 and stable for GRK6. Using bioinformatic studies we identified serines 1248 and 1291 as the major serine phosphorylation sites of the IGF-1R, and subsequent mutation analysis demonstrated clear effects on IGF-1R signaling and degradation, mirroring alterations by GRKs. Targeted mutation of S1248 recapitulates GRK2 modulation, whereas S1291 mutation resembles GRK6 effects on IGF-1R signaling/degradation, consistent with GRK isoform-specific serine phosphorylation. This study demonstrates distinct roles for GRK isoforms in IGF-1R signaling through β-arrestin binding with divergent functional outcomes.
authors.affiliation: Department of Oncology and Pathology, Karolinska Institutet and Karolinska University Hospital, 17176 Stockholm, Sweden.
authors.forename: Huiyuan
authors.initials: H
authors.lastname: Zheng
authors.forename: Claire
authors.initials: C
authors.lastname: Worrall
authors.forename: Hongchang
authors.initials: H
authors.lastname: Shen
authors.forename: Tarik
authors.initials: T
authors.lastname: Issad
authors.forename: Stefan
authors.initials: S
authors.lastname: Seregard
authors.forename: Ada
authors.initials: A
authors.lastname: Girnita
authors.forename: Leonard
authors.initials: L
authors.lastname: Girnita
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism; D054768:G-Protein-Coupled Receptor Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D017526:Receptor, IGF Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013379:Substrate Specificity; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D034741:RNA, Small Interfering; D000071557:beta-Arrestins; D012694:Serine; D017526:Receptor, IGF Type 1; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6