Deciphering the complex pharmacology of CB1: towards the understanding of a third signaling pathway

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ABSTRACT 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 Gq, G12/13 and Gs proteins. However, currently there is no evidence that Gi/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. Cannabinoid receptor CB1, an abundantly expressed GPCR that mainly couples to Gi/o, has a EAL C-motif (last three amino acids) that binds PDZ class III proteins, including PDZ-RhoGEF. Our central hypothesis is that CB1 receptor 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. 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 CB1 receptor and identification of pathways-selective ligands. We will use receptor and PDZ-RhoGEF mutations, measurements of second messengers levels (cAMP, choline and phosphatidic acid) as well as live imaging of PLD activation. 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 CB1-induced signaling; experiments are designed to characterize intracellular cascades activated by CB1-motifs (CB1-PDZ binding domain. Aim 2. Investigate the role of PDZ-RhoGEF/RhoA pathway in CB1 receptor signaling in cultured primary neurons and in vivo. The successful completion of this project will increase the current knowledge of GPCR signaling and will serve as a basis for further development of ligands selectively targeting this pathway.