The putative Gαq-inhibiting pepducin P4Pal₁₀ distinctly modulates function of the Gαi-coupled receptors FPR2 and FFA2R in neutrophils

Abstract

21 A novel mechanism of action was described, when a protease-activated receptor 4 (P4Pal 10 ) 22 derived lipopeptide (pepducin) was shown to inhibit signaling downstream of several unrelated 23 Gq-coupled receptors. We show that this putative Gq-inhibiting pepducin lacks inhibitory 24 effects on signaling downstream of the Gαq-coupled receptors for ATP (P2Y 2 R) and PAF 25 (PAFR) expressed in human neutrophils. P4Pal 10 inhibited however, signaling in neutrophils 26 activated with agonists for the Gi-coupled formyl peptide receptor 2 (FPR2) but not the 27 closely related FPR1. In addition, the P4Pal 10 pepducin was turned into an activating agonist in 28 the presence of an allosteric modulator selective for free fatty acid receptor 2 (FFA2R). The 29 results presented thus reveal Gq-independent effects of P4Pal 10 in modulating FPR2-and 30 FFA2R-mediated neutrophil activation. 31 32 Number of words: 120 33 Key words: G-protein coupled receptors (GPCRs), formyl peptide receptors (FPRs), NADPH-34 oxidase, pepducins, protease-activated receptor 4 (PAR4), neutrophils. 35 36 37 3 39 Introduction 40 41 Receptors belonging to the G-protein-coupled receptor (GPCR) family constitute a class of 42 recognition molecules involved in a broad range of physiological processes as well as 43 pathological conditions (1). The GPCRs have in common that they are polypeptides that span 44 a cell membrane seven times, and the transmembrane-spanning α-helices connect the N-45 terminal tail and three loops expressed on the cell surface, with three intracellular loops and a 46 C-terminal tail facing the cytosolic compartment (2). To mediate a cellular response following 47 recognition of an extracellular ligand that binds to its membrane exposed binding 48 cavity/domain, the cytosolic domains are structurally and functionally modified to allow and 49 mediate an intracellular coupling to a so-called heterotrimeric G-protein containing an α-and a 50 β/γ subunit (3). There are four classes of α-subunits, designated as Gαs, Gαq, Gαi/o and 51 Gα12/13, which initiate distinct as well as overlapping signaling cascades (3). The Gα subunit 52 part possesses an intrinsic GTPase activity which keeps signaling of a receptor at a very low or 53 zero level in the absence of an agonist (4). The conformational changes in the receptor, initiated 54 by the binding of an agonist, promotes an exchange of GDP for GTP, which leads to 55 dissociation and activation of the Gα subunit and a start of the G-protein dependent signaling 56 cascade (3). The large structural similarities between G-proteins makes it hard to determine the 57 precise identity of a signaling partner, and two bacterial toxins (pertussis toxin from Bordetella 58 pertussis and cholera toxin from Vibro cholera) have for long been the only tools available to 59 investigate receptor coupling to Gαi (sensitive to pertussis toxin) and Gαs (sensitive to cholera 60 toxin), respectively (5, 6). New and more efficient inhibitors of specific G-protein subunits have 61 been eagerly awaited and small...

Neutrophil Signaling That Challenges Dogmata of G Protein-Coupled Receptor Regulated Functions

“…However, P4 Pal 10 affects the function of neutrophil receptors that are Gαi-coupled; the pepducin inhibits neutrophils activated by orthosteric FPR2 agonists and activates the neutrophil FFAR2 when this is sensitized by an allosteric modulator. 100 Taken together, these results raise some questions of general character dealing not only with the molecular mechanism for specific GPCR activation by pepducins but also with recognition characteristics of FPR2 and FFAR2. It is, however, clear that not only the sequence of amino acids but also the position of charged amino acids (rather than the net charge) in a pepducin are characters of importance for function, 46,93,101 and this suggests that there are some strict structural requirements that determines if a pepducin is recognized by FPR2 or not.…”

“…It is also clear that P4 Pal 10 , the protease-activated receptor 4 derived pepducin, inhibits signaling downstream of several unrelated GPCRs having in common that they are Gαq-coupled, but it lacks inhibitory effects on signaling downstream of the Gαq-coupled receptors for ATP (P2Y 2 R) and PAF (PAFR) expressed in human neutrophils. However, P4 Pal 10 affects the function of neutrophil receptors that are Gαi-coupled; the pepducin inhibits neutrophils activated by orthosteric FPR2 agonists and activates the neutrophil FFAR2 when this is sensitized by an allosteric modulator …”