GPR97 triggers inflammatory processes in human neutrophils via a macromolecular complex upstream of PAR2 activation.

Neutrophils play essential anti-microbial and inflammatory roles in host defense, however, their activities require tight regulation as dysfunction often leads to detrimental inflammatory and autoimmune diseases. Here we show that the adhesion molecule GPR97 allosterically activates CD177-associated membrane proteinase 3 (mPR3), and in conjugation with several protein interaction partners leads to neutrophil activation in humans. Crystallographic and deletion analysis of the GPR97 extracellular region identified two independent mPR3-binding domains. Mechanistically, the efficient binding and activation of mPR3 by GPR97 requires the macromolecular CD177/GPR97/PAR2/CD16b complex and induces the activation of PAR2, a G protein-coupled receptor known for its function in inflammation. Triggering PAR2 by the upstream complex leads to strong inflammatory activation, prompting anti-microbial activities and endothelial dysfunction. The role of the complex in pathologic inflammation is underscored by the finding that both GPR97 and mPR3 are upregulated on the surface of disease-associated neutrophils. In summary, we identify a PAR2 activation mechanism that directs neutrophil activation, and thus inflammation. The PR3/CD177/GPR97/PAR2/CD16b protein complex, therefore, represents a potential therapeutic target for neutrophil-mediated inflammatory diseases.

Overexpression of FAM46A, a Non-canonical Poly(A) Polymerase, Promotes Hemin-Induced Hemoglobinization in K562 Cells.

FAM46A belongs to the FAM46 subfamily of the nucleotidyltransferase-fold superfamily and is predicted to be a non-canonical poly(A) polymerase. FAM46A has been linked to several human disorders including retinitis pigmentosa, bone abnormality, cancer, and obesity. However, its molecular and functional characteristics are largely unknown. We herein report that FAM46A is expressed in cells of the hematopoietic system and plays a role in hemin-induced hemoglobinization. FAM46A is a nucleocytoplasmic shuttle protein modified by Tyr-phosphorylation only in the cytosol, where it is closely associated with ER. On the other hand, it is located proximal to the chromatin regions of active transcription in the nucleus. FAM46A is a cell cycle-dependent poly-ubiquitinated short-lived protein degraded mostly by proteasome and its overexpression inhibits cell growth and promotes hemin-induced hemoglobinization in K562 cell. Site-directed mutagenesis experiments confirm the non-canonical poly(A) polymerase activity of FAM46A is essential for enhanced hemin-induced hemoglobinization. In summary, FAM46A is a novel poly(A) polymerase that functions as a critical intracellular modulator of hemoglobinization.

Stimulation of Vibratory Urticaria-Associated Adhesion-GPCR, EMR2/ADGRE2, Triggers the NLRP3 Inflammasome Activation Signal in Human Monocytes.

EMR2/ADGRE2 is an adhesion G protein-coupled receptor differentially expressed by human myeloid cells. It modulates diverse cellular functions of innate immune cells and a missense EMR2 variant is directly responsible for vibratory urticaria. Recently, EMR2 was found to activate NLRP3 inflammasome in monocytes via interaction with FHR1, a regulatory protein of complement Factor H. However, the functional involvement of EMR2 activation and its signaling mechanisms in eliciting NLRP3 inflammasome activation remain elusive. In this study, we show that EMR2-mediated signaling plays a critical role in triggering the activation (2nd) signal for the NLRP3 inflammasome in both THP-1 monocytic cell line and primary monocytes. Stimulation of EMR2 by its agonistic 2A1 monoclonal antibody elicits a Gα16-dependent PLC-ß activation pathway, inducing the activity of downstream Akt, MAPK, NF-κB, and Ca2+ mobilization, eventually leading to K+ efflux. These results identify EMR2 and its associated signaling intermediates as potential intervention targets of NLRP3 inflammasome activation in inflammatory disorders.

The Adhesion G Protein-Coupled Receptor GPR97/ADGRG3 Is Expressed in Human Granulocytes and Triggers Antimicrobial Effector Functions.

The adhesion family of G protein-coupled receptors (aGPCRs) comprises 33 members in human, several of which are distinctly expressed and functionally involved in polymorphonuclear cells (PMNs). As former work indicated the possible presence of the aGPCR GPR97 in granulocytes, we studied its cellular distribution, molecular structure, signal transduction, and biological function in PMNs. RNA sequencing and mass-spectrometry revealed abundant RNA and protein expression of ADGRG3/GPR97 in granulocyte precursors and terminally differentiated neutrophilic, eosinophilic, and basophilic granulocytes. Using a newly generated GPR97-specific monoclonal antibody, we confirmed that endogenous GPR97 is a proteolytically processed, dichotomous, N-glycosylated receptor. GPR97 was detected in tissue-infiltrating PMNs and upregulated during systemic inflammation. Antibody ligation of GPR97 increased neutrophil reactive oxygen species production and proteolytic enzyme activity, which is accompanied by an increase in mitogen-activated protein kinases and IκBα phosphorylation. In-depth analysis of the GPR97 signaling cascade revealed a possible switch from basal Gαs/cAMP-mediated signal transduction to a Gαi-induced reduction in cAMP levels upon mutation-induced activation of the receptor, in combination with an increase in downstream effectors of Gßγ, such as SRE and NF-κB. Finally, ligation of GPR97 increased the bacteria uptake and killing activity of neutrophils. We conclude that the specific presence of GPR97 regulates antimicrobial activity in human granulocytes.

Activation of Adhesion GPCR EMR2/ADGRE2 Induces Macrophage Differentiation and Inflammatory Responses via Gα16/Akt/MAPK/NF-κB Signaling Pathways.

EMR2/ADGRE2 is a human myeloid-restricted adhesion G protein-coupled receptor critically implicated in vibratory urticaria, a rare type of allergy caused by vibration-induced mast cell activation. In addition, EMR2 is also highly expressed by monocyte/macrophages and has been linked to neutrophil migration and activation. Despite these findings, little is known of EMR2-mediated signaling and its role in myeloid biology. In this report, we show that activation of EMR2 via a receptor-specific monoclonal antibody promotes the differentiation of human THP-1 monocytic cell line and induces the expression of pro-inflammatory mediators, including IL-8, TNF-α, and MMP-9. Using specific signaling inhibitors and siRNA knockdowns, biochemical and functional analyses reveal that the EMR2-mediated signaling is initiated by Gα16, followed by the subsequent activation of Akt, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and nuclear factor kappa-light-chain-enhancer of activated B cells. Our results demonstrate a functional role for EMR2 in the differentiation and inflammatory activation of human monocytic cells and provide potential targets for myeloid cell-mediated inflammatory disorders.