SUMMARY Understanding the mechanisms underlying GPCR signaling is crucial in order to fully comprehend their role in physiology and pathophysiology. In addition to canonical second messengers (cAMP, cGMP and IP3) and β- arrestin signaling, small GTPase proteins, such as Rho GTPases are largely involved in GPCR-mediated signal transduction. Guanine nucleotide exchange factors (GEFs) convert Rho GTPases from an inactive (GDP-bound) state to an active state (GTP-bound). Rho-GEFs can be activated by Gq, G12/13 and Gs proteins. However, currently there is no evidence that Gi/0-WT can directly activate RhoGEFs. PDZ domains are structural protein domains that recognize simple linear amino acid motifs often at the protein C-terminal (C-motif). RhoA, activated by PDZ-RhoGEFs, has important signaling roles, by activating phospholipase D (PLD) and transcription factors. Kappa opioid receptor (KOR), an abundantly expressed GPCR that mainly couples to Gi/o, has a KPV C-motif (last three amino acids) that binds PDZ class III proteins, including PDZ-RhoGEF. Our central hypothesis is that KOR activation, in addition to engaging cAMP inhibition and β-arrestin pathways, initiates an additional signaling mechanism downstream to PDZ-RhoGEF leading to activation of RhoA and subsequent activation of PLD. Activation of PLD generates two distinct second messengers, phosphatidic acid, which activates the mTOR pathway, and choline, which activates Sigma1 receptors. In addition to in vitro studies, this pathway that plays an essential role in KOR signaling will be studied in vivo on antipruritic effects of KOR agonists. Antipruritic effects of KOR agonists are well established and as of today, there is one FDA-approved KOR agonist (difelikefalin) for the treatment of chronic itch of chronic kidney diseases. We will use a multidisciplinary approach, combining state-of-the-art molecular and pharmacological approaches for a comprehensive investigation of the signaling pathways elicited by activation of C-motifs of KOR. Receptor and PDZ-RhoGEF mutations, measurements of second messenger levels (cAMP, choline and phosphatidic acid) as well as live imaging of PLD activation will be used. Acute itch model of scratching will be used for in vivo studies. We provide solid preliminary results supporting the feasibility of the project and the ability of our team to complete the work proposed. The project has two aims: Aim 1. Investigate the role of PDZ-binding domain in KOR-induced signaling; experiments are designed to characterize intracellular cascades activated by KOR-motifs (KOR-PDZ binding domain). Aim 2. Investigate the role of PDZ-RhoGEF/RhoA pathway in antipruritic effects of KOR agonists. RoA and sigma 1 receptor antagonists will be tested on scratch inhibiting effects of three different KOR agonists (U50,488 as prototype, nalbuphine, and difelikefalin as peripherally restricted). The successful completion of this project will increase the current knowledge of GPCR sign...