Specific GPCRs Elicit Unique Extracellular Vesicle MiRNA Array Signatures: An Exploratory Study

3.All cells secrete extracellular vesicles (EVs) containing nucleic acid cargo, including microRNAs (miRNAs), that regulate the function of receiving cells. G protein-coupled receptors (GPCRs) affect intracellular function via multiple signaling cascades. However, the mechanisms of GPCR intercellular signaling through EV miRNA activity are unknown. Human U2 osteosarcoma cells expressing native GPCRs were used to selectively stimulate distinct G protein signaling cascades (Gi, Gq, G12/13, and {beta}-arrestin) by members of specific receptor subclasses including the adenosine receptor A1 (ADORA1), the histamine receptor H1 (HRH1), the frizzled class receptor 4 (FZD4), and the atypical chemokine receptor 3 (ACKR3), respectively. We hypothesized that stimulation of specific classes of GPCRs would cause the release of EVs containing miRNAs with receptor-specific up- or down-regulated expression, affecting unique pathological downstream signaling cascades. Receptor-specific agonists dose-dependently increased respective signaling cascade intermediates. We found no change in the quantity of EVs ([~]200nm diameter), but there were distinct EV miRNA signatures following stimulation of GPCRs. Network analyses of differentially expressed miRNA and their predicted targets validated the linkage between specific receptors and cell function and pathological states. The data can be used to reverse engineer mechanisms involving EV miRNAs for various physiological and pathological processes. GPCRs are major pharmacological targets, so understanding the mechanisms that stimulate or inhibit GPCR-mediated changes in extracellular miRNA signatures could improve long- and short-term therapeutic and unwanted drug effects. SummaryStimulation of specific GPCRs elicited unique EV miRNA signatures that up- and down-regulated predicted biological and pathological cellular processes. 4. Significance StatementG protein-coupled receptors (GPCR) are therapeutic targets for various human diseases. However, the mechanisms of GPCR signaling through extracellular vesicle (EV) miRNA activity are unknown. Here, we identified unique EV miRNA signatures resulting from GPCR activation. The miRNA functional analyses indicate that the downstream signaling effects and their functional networks differ by receptor. This study has important implications for developing new drugs and their long-term and "off-target" effects. 5. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC="FIGDIR/small/659918v2_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1a9812org.highwire.dtl.DTLVardef@1f5cbf3org.highwire.dtl.DTLVardef@20d1fdorg.highwire.dtl.DTLVardef@ba4fad_HPS_FORMAT_FIGEXP M_FIG C_FIG