Complement-activation fragment C4a mediates effector functions by binding as untethered agonist to protease-activated receptors 1 and 4 HongBin Wanga, Daniel Ricklina,b,1, and John D. Lambrisa,1 aDepartment of Pathology & Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104; and bDepartment of Pharmaceutical Sciences, University of Basel, 4056 Basel, Switzerland Edited by Douglas T. Fearon, Cornell University, Cambridge, United Kingdom, and approved August 28, 2017 (received for review May 5, 2017) C4a is a small protein released from complement component C4 but not the human receptor (7). It has to be noted that these early upon activation of the complement system’s classical and lectin studies were typically performed with plasma-purified C4a, which pathways, which are important constituents of innate immune sur- may have contained small but physiologically relevant amounts of veillance. Despite the structural similarity between C4a and well- other anaphylatoxins. Recent studies using recombinant human described anaphylatoxins C3a and C5a, the binding partner and C4a have shown that the protein can inhibit C3a- or C5a-mediated biological function of C4a have remained elusive. Using a cell-based chemoattractant and secretagogue functions in mast cells, impair reporter assay, we screened C4a against a panel of both known and C5a-induced neointima formation, and protect against hyperoxic orphan G protein-coupled receptors and now provide evidence that lung injury via a macrophage-dependent signaling pathway (8–10). C4a is a ligand for protease-activated receptor (PAR)1 and PAR4. Whereas these results may indicate a potential competition of C4a Whereas C4a showed no activity toward known anaphylatoxin re- for the anaphylatoxin receptors, direct binding could not be con- ceptors, it acted as an agonist for both PAR1 and PAR4 with nano- firmed to any of the receptors. Several studies have suggested that molar activity. In human endothelial cells, ERK activation by C4a was the receptor for C4a is distinct from C3aR, C5aR1, or C5aR2 (6, 7, α mediated through both PAR1 and PAR4 in a G i-independent signal- 11) but no suitable candidate has been identified. ing pathway. Like other PAR1 activators, C4a induced calcium mobi- Nevertheless, the strong homology of C4a to C3a and C5a, INFLAMMATION α β IMMUNOLOGY AND lization through the PAR1/G q/PLC signaling axis. Moreover, C4a which both bind G protein (C3aR, C5aR1)- or β-arrestin (C5aR2)- increased stress fiber formation and enhanced endothelial perme- coupled seven transmembrane receptors (11, 12), renders this class ability, both of which were reduced by PAR1 antagonists. In sum, of receptors a likely target for C4a. Indeed, a recent study has our study identifies C4a as an untethered agonist for PAR1 and demonstrated that C4a can decrease intracellular cAMP levels by PAR4 with effects on cellular activation and endothelial permeability, acting on Gi protein-coupled pathways (9). Thus, we hypothesized thereby revealing another instance of cross-talk between the com- that the biological function of C4a is not mediated through ana- plement system and other host defense pathways. phylatoxin receptors, but instead through a hitherto-unidentified G protein-coupled receptor (GPCR). To test this hypothesis, we used complement | C4a | PAR1 | PAR4 | endothelial cells a GPCR reporter assay, which revealed that C4a is a specific ligand for protease-activated receptors 1 and 4 (PAR1 and PAR4, re- he host defense relies on a tight interplay and extensive cross- spectively). These receptors exert various functions on platelets and Ttalk between innate immune pathways such as the comple- ECs, among others, and are traditionally activated by cleavage of a ment system, the contact and coagulation cascades, and cellular tethered ligand by proteases such as thrombin (13). In the case of components, including platelets and endothelial cells (ECs). C4a, however, PAR1/PAR4 activation appeared to be mediated by Although typically conferring protection from microbial in- truders and accumulating debris, host defense systems can Significance contribute to various thromboinflammatory, acute-phase, and age- related disorders if inappropriately triggered (1). Complement plays a key role in these processes by sensing pathogen- and How C4a transduces signaling and generates various biological damage-associated molecular patterns and initiating a cascade functions has long been unresolved. Using a cell-based re- that facilitates the elimination of invading cells via phagocytosis porter assay, we screened C4a against a panel of 168 known and lytic damage and releasing inflammatory mediators to prop- and 73 orphan G protein-coupled receptors and now provide agate danger signaling (1, 2). The anaphylatoxins C3a and C5a, evidence that C4a is a ligand for protease-activated receptor which are released upon activation of complement components (PAR)1 and PAR4. In human endothelial cells, our assessment of C3 and C5, respectively, are among the most potent complement- ERK activation and calcium mobilization in the presence of derived effectors that exert a number of well-described functions various antagonists and inhibitors suggests that C4a-mediated through binding to the anaphylatoxin receptors C3aR, C5aR1, cell activation is mediated through PAR1 and PAR4. Our results and/or C5aR2 (3). demonstrate that C4a is a nontraditional agonist for PAR1 and Although complement activation through the classical and PAR4 whose direct functional effects indicate a potentially lectin pathways leads to the release of an ∼10-kDa protein from significant direct linkage between the complement, coagulation, component C4, termed C4a, that shares high homology with the and endothelial barrier systems. anaphylatoxins, the receptor and biological functions of this ac- Author contributions: H.W., D.R., and J.D.L. designed research; H.W. and D.R. performed tivation fragment have thus far remained elusive (4). Previously, ·− research; H.W. and D.R. contributed new reagents/analytic tools; H.W., D.R., and J.D.L. C4a had been reported to inhibit C3a-induced O2 generation in analyzed data; and H.W., D.R., and J.D.L. wrote the paper. guinea pig macrophages, induce contraction of guinea pig ileum, The authors declare no conflict of interest. and produce immediate erythema/edema when injected into This article is a PNAS Direct Submission. human skin (5, 6). Whereas its ability to desensitize the action of 1To whom correspondence may be addressed. Email: [email protected] or lambris@ C3a-induced contraction of guinea pig ileum suggested a func- upenn.edu. tion for C4a closely related to C3a (5), it was later shown that This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. human C4a may act as a potent agonist of the guinea pig C3aR 1073/pnas.1707364114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1707364114 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 direct binding to the receptor. These unexpected findings were A F2R-CHO-K1 (PAR1) B F2R-CHO-K1(PAR1) corroborated in various cell-based assays that allowed us to deduce 10 C4a TFLLR-NH 5 the signaling pathways involved and describe C4a’s functional ef- 2 C4a+RWJ56110 0.3µM AY-NH2 C4a+RWJ56110 1 µM fects on EC permeability. Taken together, our results demonstrate 8 C3a 4 C4a+RWJ56110 10 µM C5a ) that C4a is a nontraditional agonist for PAR1 and PAR4 with direct 5 C4a functional effects and potential implications as a direct link between 6 3 the complement, coagulation, and endothelial barrier systems. 4 2 Results (x 10 RLU 2 1 Human C4a Is a Ligand for PAR1 and PAR4. Previous studies have control of change Fold suggested that human C4a transduces signaling through a hitherto 0 0 unknown receptor, likely of the GPCR superfamily, to exert its -10 -8 -6 -4 -10 -8 -6 -4 proposed biological functions (6, 7, 9, 11). In an attempt to iden- log[Agonist], M log[C4a], M tify potential C4a-reactive GPCR proteins, we screened plasma- derived human C4a in a cell-based assay comprising a panel of C F2RL3-CHO-K1 (PAR4) D F2RL3-CHO-K1 (PAR4) 9 5 C4a 168 known (gpcrMAX) and 73 orphan (orphanMAX) human TFLLR-NH 2 C4a+tcY-NH2 10 µM GPCRs. This assay employs engineered GPCR-expressing cell lines AY-NH C4a+tcY-NH2 100 µM 2 4 C4a+tcY-NH 300 µM and a reporter system based on β-arrestin recruitment and enzyme C3a 2 C5a ) fragment complementation to monitor agonistic or antagonistic 6 5 C4a 3 activities (14) (Fig. S1A). In agonist mode, screening of the gpcrMAX panel revealed two GPCRs that met the selectivity 2 3 criteria as putative targets for C4a (Fig. 1 and Table S1): PAR1 (x 10 RLU (F2R) and PAR4 (F2RL3). We chose a screening concentra- 1 tion of 600 nM C4a, which corresponds to an activation of control of change Fold ∼20% of the total plasma pool of complement component C4. 0 0 -10 -8 -6 At this concentration, C4a was able to induce 53% and 97% of -10 -8 -6 -4 log[C4a], M the maximal control ligand response for PAR1 and PAR4, re- log[Agonist], M spectively. Importantly, C4a did not elicit any activation of the Fig. 2. C4a acts as an agonist for PAR1 and PAR4. CHO-K1 cells expressing anaphylatoxin receptors (C3aR, C5aR1, and C5aR2) or other either PAR1 or PAR4 were seeded in a 96-well plate and stimulated with members of the PAR family (PAR2 and PAR3), confirming PAR1 agonist TFLLR-NH2, PAR4 agonist AY-NH2, human C4a, C3a, or C5a. After that it is a specific ligand for PAR1 and PAR4. No antagonistic stimulation, the chemiluminescent signal was detected. Culture medium alone activity (Fig. S1B and Table S2)ororphanGPCRactivation wasusedasacontrol.(A) C4a and PAR1 agonist TFLLR-NH2 dose-dependently (Fig. S1C and Table S3) for C4a was detected. activate PAR1.