The goal of our research is to understand the cellular and molecular mechanisms of histamine-independent itch. Primary sensory neurons in dorsal root ganglia (DRG) play an essential role in generating itch by detecting itch stimuli through their peripheral axons in the skin and sending the signals to the spinal cord via their central axons. The best characterized itch mediator is histamine. However, anti-histamine drugs are ineffective in most itch conditions suggesting the involvement of histamine- independent pathways. The major hurdle in understanding histamine-independent itch is the dearth of molecular markers that label itch-sensitive neurons in DRG and cell surface receptors directly activated by itch stimuli other than histamine. Recently, we have shown that Mrgprs, a family of G protein-coupled receptors specifically expressed in a small subset of DRG neurons, function as receptors for the anti- malaria drug chloroquine and are required for chloroquine-induced itch. Besides chloroquine, Mrgprs and Mrgpr-expressing DRG neurons also respond to several other itch-inducing compounds suggesting that Mrgprs are novel itch receptors by directly sensing these compounds and that neurons expressing these receptors transduce itch signals. In this proposal, we will take molecular, genetic, behavioral, and electrophysiological approaches to dissect the functions of Mrgprs and properties of Mrgpr-expressing DRG neurons in itch. Our preliminary data show that Mrgprs mediate itch induced by SLIGRL, a peptide agonist for protease-activated receptor 2 (PAR2). This surprising result challenges the traditional notion that PAR2 functions as the itch receptor in DRG to mediate protease-induced itch. Aim I is to test the hypothesis that Mrgprs function as receptors for the cleaved N-terminus of PAR2 generated by proteases in itch signaling. We have recently identified a small molecule compound that can specifically inhibit human and mouse Mrgpr activation by several itch-inducing compounds including chloroquine and SLIGRL in heterologous cells. In Aim II, we will determine whether treatment with the antagonist can block chloroquine- and SLIGRL-induced neuronal and behavioral responses in mice. Our cellular analyses suggest that Mrgpr-expressing neurons are itch-sensitive neurons. In Aim III, we will use our newly generated transgenic mouse lines in which Mrgpr-expressing DRG neurons are labeled by GFP-Cre to study the electrophysiological properties and axonal projections of these neurons in the periphery and the spinal cord. Functional analysis of Mrgprs and Mrgpr-expressing neurons will provide insight into key mechanisms of itch as well as open the door for the development of novel itch therapeutics.