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

Abstract

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.

Structural basis of GAIN domain autoproteolysis and cleavage-resistance in the adhesion G-protein coupled receptors

“…Crystal structures of the GAIN domains of human ADGRB3/BAI3 (B3) and rat ADGRL1/LPHN1 (L1) revealed a fold comprising an α-helical subdomain A followed by the C-terminal subdomain B, which consists of a twisted β-sandwich including eleven β-strands with additional short α-helices and β-strands located within loop regions 4 . Additional crystal structures of murine ADGRG1/GPR56 (G1) 5 , zebrafish ADGRG6/GPR126 (G6) 6 , human AD-GRG3/GPR97 (G3) 7 and ADGRF1/GPR110 (F1) 8 confirmed the overall architecture of the GAIN domain fold. Proteolytic cleavage of aGPCRs was first indicated for human ADGRE5/CD97, which is processed into two noncovalently associated fragments 9 .…”

“…Even though it was shown for L1, G1, G3, and G6 that the GAIN domain is sufficient for self-cleavage 4–7 , a possible cause of the lack of GPS cleavage for the ectodomain constructs of B2 may be the lack of cleavagemodulating factors. For example, we surmised that the presence of the 7TM domain may aid in activation of the GAIN domain for the autoproteolysis reaction.…”

“…We could crystallize the HormR-GAIN domains of ADGRB2 and determined the crystal structure of this aGPCR GAIN domain in the uncleaved state. After structure determination of the HormR-GAIN domains of ADGRB3 4 , this is the second structure of an uncleaved aGPCR GAIN domain, whereas four structures are available for GPS-cleaved GAIN domains (L1, G1, G3, G6) 4–7 . A comparison of the B2 and B3 GAIN domains with AlphaFold models of cleavage-competent aGPCR indicated that the fold of the GPS sequence is rather similar and that the immediate environment of the GPS in the GPS-active receptors does not contain any conserved residues besides the GPS sequence itself, that are capable of acid-base catalysis, which would be a typical situation for a proteolytic enzyme.…”

“…ADGRG5 contains an HLT motif a the GPS, is cleavage-incompetent upon expression in COS-7 cells, but exhibits Stachel -mediated GPCR signaling 24 . While the GAIN domain structures of L1 4 , G1 5 , G3 7 and G6 6 were determined in the cleaved state, B3 was obtained in an uncleaved state 4 . However, B3 contains an RLS motif at the GPS and an arginine side chain should not be able to act as a base and deprotonate the serine nucleophile for autoproteolysis 12 (Fig.…”

Role of Adhesion G Protein-Coupled Receptors in Immune Dysfunction and Disorder

“…The role of the GPR97-associated receptor complex in pathologic inflammation was realized by upregulated GPR97 and mPR3 expression on the surface of neutrophils in various inflammatory diseases, including appendicitis, bacterial sepsis, and granulomatosis with polyangiitis (GPA). Our results hence reveal a novel aGPCR-GPCR transactivation mechanism in human neutrophils that directs inflammatory responses [ 153 ].…”