SLIGRL is a widely used pruritogen for studying histamine-independent itch (14
). Here, we show that Mrgprs (likely MrgprC11), not PAR2 as previously thought, are receptors that mediate this type of itch in mice. Our loss-of-function analyses indicate that Mrgprs, rather than PAR2, are required for behavioral and neuronal responses induced by SLIGRL. In line with these findings, SLIGRL directly activates only ~3 to 4% of total DRG neurons, which is consistent with the restricted presence of Mrgprs, particularly MrgprC11, in DRG (8
). Moreover, all SLIGRL-sensitive neurons also respond to BAM(8–22), a specific agonist for MrgprC11 (8
). Like SLIGRL, BAM(8–22) induces an Mrgpr-dependent scratching response (8
). These data further support the notion that Mrgprs are a family of GPCRs that play essential roles in itch responses by directly sensing different pruritogens.
Because MrgprC11 can be activated by many peptides with common C-terminal motifs, such as -RFamide, -RYamide, or -RYG (11
), it is not completely surprising that SLIGRL is also an agonist forMrgprC11 among the 12 Mrgprs we tested. SLIGRL ends with -RLamide, and removing the leucine or amidation at the C terminus (for example, SLIGR or SLIGRL without NH2
) abolishes its ability to activate MrgprC11. These data suggest that MrgprC11 peptide agonists need arginine at the second to last position and amidation at their C termini. Although SLIGR can activate PAR2 at an EC50
comparable to that of SLIGRL as monitored by [Ca2+
increase in heterologous cells, it failed to directly induce any calcium response in wild-type DRG neurons where PAR2 is present. This discrepancy may be due to high PAR2 expression in the heterologous system, which results in coupling to the downstream calcium pathway. Our results with SLIGR, along with previous findings, suggest that PAR2 signaling in DRG neurons positively modulates TRPV1 activity mainly through protein kinase Cε (PKCε) and PKA pathways (19
). Such modulation induces thermal hyperalgesia in a PAR2-dependent manner. Because SLIGR, which activates PAR2 but not MrgprC11, failed to evoke any scratching, it is unlikely that PAR2 plays a major role in itch by directly sensing the peptide in DRG neurons ().Our results also suggest that trypsin-induced itch and thermal hyperalgesia are mediated by an unknown mechanism that is independent of PAR2 or Mrgprs, for example, through other PARs ().
Although our data suggest that SLIGRL-induced scratching is mediated by MrgprC11, not PAR2, they do not diminish the roles of PAR2 in histamine-independent itch but broaden our knowledge of the complex roles of GPCRs in the physiology of pruritus. It is possible that itch induced by other PAR2 activators such as tryptase is mediated by PAR2 in humans (5
). In addition, species differences in itch signaling may also explain the difference between our current findings in mice and activation of PAR2 observed in human pruritic responses. Further experiments using PAR2−/−
mice, serine protease–deficient mice, and “humanized” PAR2 knock-in mice in skin disease models should address these issues. Previous studies have suggested that the activation mechanisms of PAR2 by peptides and proteases are likely to be different (20
). Our study not only reveals a previously unappreciated role of MrgprC11 in peptide-induced itch but also emphasizes the differences between protease- and peptide-mediated itch signaling.
The role of MrgprC11 in SLIGRL-induced itch raises a possible interaction between PAR2 and MrgprC11, which may contribute to the physiological relevance of our finding. It is possible that soluble SLIGRL peptide is released from PAR2 by cleaving at the C terminus of SLIGRL sequence, which is a cleavage site for chymotrypsin on the basis of a computer program search for protease cleavage sites (21
). Because both PAR2 and MrgprC11 are present in DRG neurons, it is conceivable that soluble SLIGRL can activate nearby MrgprC11. Indeed, intermolecular interaction between two PARs has been reported (22
). Future experiments are needed to test the potential interaction between PAR2 and MrgprC11 in vivo. Detailed genetic and molecular information about the roles of GPCRs in DRG neuron function will provide insight for developing novel anti-pain and anti-itch drugs.