Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells

Citation: Subramanian H, Gupta K, Ali H ( Roles for NHERF1 and NHERF2 on the Regulation of C3a Receptor Signaling in Human Mast Cells Hariharan Subramanian 0 Kshitij Gupta 0 Hydar Ali 0 Roland Seifert, Medical School of Hannover, United States of America 0 Department of Pathology, School of Dental Medicine, University of Pennsylvania , Philadelphia, Pennsylvania , United States of America Background: The anaphylatoxin C3a binds to the G protein coupled receptor (GPCR, C3aR) and activates divergent signaling pathways to induce degranulation and cytokine production in human mast cells. Adapter proteins such as the Na+/H+ exchange regulatory factor (NHERF1 and NHERF2) have been implicated in regulating functions of certain GPCRs by binding to the class I PDZ (PSD-95/Dlg/Zo1) motifs present on their cytoplasmic tails. Although C3aR possesses a class I PDZ motif, the possibility that it interacts with NHERF proteins to modulate signaling in human mast cells has not been determined. Methodology/Principal Findings: Using reverse transcription PCR and Western blotting, we found that NHERF1 and NHERF2 are expressed in human mast cell lines (HMC-1, LAD2) and CD34+-derived primary human mast cells. Surprisingly, however, C3aR did not associate with these adapter proteins. To assess the roles of NHERFs on signaling downstream of C3aR, we used lentiviral shRNA to stably knockdown the expression of these proteins in human mast cells. Silencing the expression of NHERF1 and NHERF2 had no effect on C3aR desensitization, agonist-induced receptor internalization, ERK/Akt phosphorylation or chemotaxis. However, loss of NHERF1 and NHERF2 resulted in significant inhibition of C3a-induced mast cell degranulation, NF-kB activation and chemokine production. Conclusion/Significance: This study demonstrates that although C3aR possesses a class I PDZ motif, it does not associate with NHERF1 and NHERF2. Surprisingly, these proteins provide stimulatory signals for C3a-induced degranulation, NF-kB activation and chemokine generation in human mast cells. These findings reveal a new level of complexity for the functional regulation of C3aR by NHERFs in human mast cells. - Funding: This work was supported by a grant to HA from the National Institutes of Health (AI-080852) and a grant to HS from the American Heart Association (12POST9260009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Cross-linking of high affinity IgE receptors (FceRI) on mast cells is known to play an important role in allergic and hypersensitive diseases [1]. Fukuoka et al [2] showed that activation of human mast cells via FceRI results in the secretion of tryptase, which generates sufficient amounts of C3a from C3 to cause mast cell degranulation. They proposed that C3a-induced mast cell activation may play an important role in mediating allergic diseases. Indeed, Shafer et al., [3] recently demonstrated that IgEmediated passive cutaneous anaphylaxis resulted in local increase in C3a levels and that subsequent activation of C3aR on mast cells contributed to allergic skin response. Not surprisingly, we have shown that C3a causes degranulation and chemokine generation in human mast cells and in transfected RBL-2H3 cells [46]. However, the mechanisms involved in the regulation of C3aR signaling in mast cells remain poorly defined. A large number of multi-domain scaffolding proteins, including PDZ (PSD-95/Dlg/Zo1) domain containing proteins, associate with GPCRs [7,8]. There are three general classes of PDZ domains; class I domain, which recognize the carboxyl terminal motif S-T-X-W, (where W indicates hydrophobic amino acid and X indicates any amino acid), class II domain which recognize carboxyl terminal motif W-X-W and class III domains, which recognize D/E-X-W as their preferred carboxyl terminal motif [9]. Na+/H+ exchanger regulatory factor-1 and 2 (NHERF1, EBP-50, SLC9A3R1 and NHERF2, TKA-1, SLC9A3R1) are two class I PDZ domain adapter proteins that bind to several GPCRs to regulate their signaling [10]. Both NHERF1 and NHERF2 have been shown to bind to parathyroid hormone receptor (PTHR) and b2-adrenergic receptor (b2-AR) via their PDZ binding domains to modulate receptor desensitization and internalization [1115]. NHERF proteins can also regulate GPCR signaling in receptor-independent manner via their association with downstream signaling proteins. For example, NHERF1 and NHERF2 associate with phospholipase C-b to regulate GPCR activation [16,17]. Moreover, NHERF1 blocks PTH-induced ERK1/2 phosphorylation downstream of PKA via a receptorindependent but Akt-dependent pathway [18]. C3aR is one of the few GPCRs expressed in human mast cells that possess a class I PDZ motif. We therefore postulated that NHERF1 or NHERF2 could associate with C3aR to modulate signaling in mast cells. We have recently utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of receptors, protein kinases and adapter molecules in human mast cell lines, HMC-1, LAD2 and primary CD34+-derived human mast cells to study receptor signaling [1921]. We used a similar approach for the current study and used both human mast cells lines (HMC-1 and LAD2) as well primary human CD34+-derived mast cells and show that although NHERF1 and NHERF2 do not interact with C3aR, they play a critical role in modulating C3a-induced degranulation and chemokine production. NHERF1 and NHERF2 are expressed in human mast cells but they do not interact with C3aR NHERF proteins are highly expressed in epithelial cells of the gastrointestinal tract and airways [10,22,23]. Since the goal of the present study was to determine the role of NHERFs on regulating C3aR signaling in mast cells, we wanted to first test if these proteins are expressed in human mast cells. We therefore performed reverse transcription PCR on cDNAs obtained from an immature (HMC-1), mature (LAD2), and primary (CD34+derived) human mast cells using primers specific for human NHERF1 and NHERF2. Fig. 1A shows that mRNAs for both NHERF1 and NHERF2 were detected in all three types of human mast cells. To determine if NHERFs physically associate with C3aR, we co-transfected HA-tagged C3aR and Flag-tagged NHERF1 or NHERF2 in HEK 293 cells and performed coimmunoprecipitation experiments. Surprisingly, NHERF1 or NHERF2 failed to interact with C3aR even when the cells were stimulated with C3a (Fig. 1B and C, lane 3, upper panel). It is quite possible that the lack of C3aR-NHERF interaction reflects the experimental condition used for the present study. It has previously been shown that NHERF1 and NHERF2 strongly associated with b2-adrenergic receptor (b2-AR) [11,24]. We therefore co-expressed HA-tagged b2-AR and Flag-tagged NHERF1 or NHERF2 and performed co-immunoprecipitation study under the exact same condition as was done for C3aRNHE (...truncated)