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Item Human GPR17 missense variants identified in metabolic disease patients have distinct downstream signaling profiles(Elsevier, 2021-07) Conley, Jason M.; Sun, Hongmao; Ayers, Kristin L.; Zhu, Hu; Chen, Rong; Shen, Min; Hall, Matthew D.; Ren, Hongxia; Pediatrics, School of MedicineGPR17 is a G-protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Such evidence is primarily drawn from mouse knockout studies and suggests GPR17 as a potential novel therapeutic target for the treatment of metabolic diseases. However, links between human GPR17 genetic variants, downstream cellular signaling, and metabolic diseases have yet to be reported. Here, we analyzed GPR17 coding sequences from control and disease cohorts consisting of individuals with adverse clinical metabolic deficits including severe insulin resistance, hypercholesterolemia, and obesity. We identified 18 nonsynonymous GPR17 variants, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, membrane localization, and downstream signaling profiles of nine GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). These nine GPR17 variants had similar protein expression and subcellular localization as wild-type GPR17; however, they showed diverse downstream signaling profiles. GPR17-G136S lost the capacity for agonist-mediated cAMP, Ca2+, and β-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT, but showed impaired Ca2+ and β-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca2+ signaling, but unaffected agonist-stimulated β-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with diverse signaling profiles, including differential signaling perturbations that resulted in GPCR signaling bias. Our findings provide a framework for structure-function relationship studies of GPR17 signaling and metabolic disease.Item Structural Basis of Arrestin Binding to Cell Membranes(2024-04) Miller, Kyle Warren; Chen, Qiuyan; Takagi, Yuichiro; Georgiadis, Millie M.; Hurley, Thomas D.Two non-visual arrestins, arrestin2 (Arr2) and arrestin3 (Arr3), selectively interact with activated and phosphorylated G protein-coupled receptors (GPCRs) and play crucial roles in regulating many important physiological processes. Arrestins also engage the lipid bilayer surrounding activated GPCRs, which further potentiates arrestin activation and regulates GPCR trafficking in cells. Because of this, structural and functional understanding of arrestins would provide insight in enhancing arrestin’s GPCR desensitization for various diseases where constitutively active GPCR mutants play a role including congenital endocrine disorders and familial gestational hyperthyroidism. To better understand the membrane binding role of arrestins, we performed in vitro binding assays and demonstrated that Arr2 selectively binds to nanodiscs containing Phosphatidylinositol 4,5-bisphosphate (PIP2) even in the absence of different binding sites. Our cryo-electron microscopy (Cryo-EM) structure of Arr2 in complex with PIP2 nanodisc reveals that multiple structural elements of Arr2, including the finger loop, C domain and C-edge loop, contribute to membrane binding. Eliminating one individual site does not significantly impact Arr2 binding to the nanodisc. Moreover, a preactivated variant of Arr2 shows increased binding to the nanodisc than wildtype. We also labeled four potential membrane binding sites with monobromobimane (mBrB) and detected different levels of fluorescence increase in the presence of nanodisc containing various types of phospholipids. Overall, our study provides detailed structural evidence on how arrestins engage the membrane via multiple contact points and how this can impact arrestin-mediated signaling.