A pepducin designed to modulate P2Y 2 R function interacts with FPR2 in human neutrophils and transfers ATP to an NADPH-oxidase-activating ligand through a receptor cross-talk mechanism

Abstract

Several G-protein-coupled receptors (GPCRs) can be activated or inhibited in a specific manner by membrane-permeable pepducins, which are short palmitoylated peptides with amino acid sequences identical to an intracellular domain of the receptor to be targeted. Unlike the endogenous P2Y2R agonist ATP, the P2Y2PalIC2 pepducin, which has an amino acid sequence corresponding to the second intracellular loop of the human ATP receptor (P2Y2R), activated the superoxide anion-generating NADPH-oxidase in neutrophils. In addition to having a direct effect on neutrophils, the P2Y2R pepducin converted naïve neutrophils to a primed state, which secondarily responded to ATP by producing superoxide. A pepducin with a peptide identical to the third intracellular loop of P2Y2R (P2Y2PalIC3) exhibited the same basic functions as P2Y2PalIC2, whereas one with a peptide that was identical to the first intracellular loop (P2Y2PalIC1) lacked these functions. The responses induced in neutrophils by the P2Y2R pepducins were not inhibited by the P2Y2R antagonist AR-C118925, and the receptor desensitization profile suggested the involvement of FPR2 rather than P2Y2R. Accordingly, antagonists/inhibitors of FPR2 attenuated the activities of the P2Y2R pepducins, which also selectively activated FPR2-overexpressing cells. In summary, we show that pepducins supposed to target P2Y2R activate human neutrophils through FPR2. We also show that the P2Y2PalIC2 pepducin can convert ATP from a non-activating agent to a potent neutrophil NADPH-oxidase activator. The molecular basis of this phenomenon involves cross-talk between the receptor/ligand pairs of P2Y2R/ATP and FPR2/P2Y2-pepducin.

Formyl peptide derived lipopeptides disclose differences between the receptors in mouse and men and call the pepducin concept in question

“…Even if it is hard to understand the basic mechanism for how the peptide part of a pepducin translocates to the inner leaflet of the membrane and how two identical peptide sequences interact and by that either inhibits or activates receptor signaling, the model creates some in-built restrictions, i) a pepducin should only activate/inhibit the function of receptors that contains an identical sequence in one of the intracellular signaling domains, and ii) conventional antagonists that block the orthosteric binding site of the receptor at the extracellular surface should not affect the activity induced by a receptor activating pepducin. The data presented in this study together with our earlier studies are not always consistent with these restrictions [ 24 , 25 ]. Several FPR2 activating pepducins are sensitive to conventional FPR2 antagonists and likewise, binding experiments reveal that conventional agonists compete with a pepducin for receptor binding [ 20 ].…”

“…The FPR/Fpr pepducins are receptor-selective although the functional link (activating or inhibiting) between the targeted receptor and the peptide sequences of the pepducins is missing. In contrast to FPR2/Fpr2 pepducins, FPR1- and Fpr1-derived pepducins designed from their respective third intracellular loops lack FPR1/Fpr1 modulating effects ([ 24 ], this study). The close sequence similarities between the third intracellular loops in FPR1 and FPR2 ( Table 1 ) could possibly explain why the FPR1 pepducin targets also FPR2.…”

Reactivation of Gαi-coupled formyl peptide receptors is inhibited by Gαq-selective inhibitors when induced by signals generated by the platelet-activating factor receptor

“…However, the functional response is inhibited by a conventional antagonist, and the binding can be outcompeted by an orthosteric agonist [18]. These data put the pepducin dogma to question, at least when it comes to FPR2, and that questioning is strongly supported by data showing that FPR2 is activated by pepducins originating from P2Y 2 R and CXCR4, receptors that have no sequence similarities to FPR2 [40,41]. Importantly, in the context of crosstalk, the PAFR-initiated crosstalk is not limited to the F2Pal 10 -desensitized receptors or the FPR2inhibitor PBP 10 ; the inhibitor could be replaced by two other FPR2 specific/selective inhibitors, the widely used WRW 4 -peptide [42] and a recently described FPR2 peptidomimetic-inhibitor Pam-(Lys-bNSpe) 6 -NH 2 [22], and the same results in crosstalk and sensitivity to the Gaq inhibitor are obtained when the F2Pal 10 pepducin is replaced with a conventional peptide agonist (the formylated peptide PSMa2 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] ).…”

Characterisation of P2Y2 receptors in human vascular endothelial cells using AR‐C118925XX, a competitive and selective P2Y2 antagonist

“…AR-C118925XX was developed by AstraZeneca around 20 years ago as a P2Y 2 antagonist, but only a conference abstract was published at the time (Meghani, 2002), which did not include crucial pharmacological properties, such as its K B or pA 2 . Several studies have since been published that used AR-C118925XX to investigate the role of native P2Y 2 receptors in the actions of P2Y agonists in various cell types (Cosentino et al, 2012;Gabl et al, 2016;Hochhauser et al, 2013;Kemp, Sugar, & Jackson, 2004;Magni, Merli, Verderio, Abbracchio, & Ceruti, 2015;Onnheim et al, 2014;Wang et al, 2015; see also review by Rafehi & Müller, 2018). They did not, however, report the K B or pA 2 of AR-C118925XX or relate the concentrations used (mostly 1 and 10 μM) to its potency or selectivity.…”