Cannabinoid Receptors and Associated Proteins-Renewal

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Project Summary The CB1 cannabinoid receptor (CB1R) regulates neuronal processes critical for retrograde signaling to reduce excessive neurotransmitter release, neuronal development in fetal and adolescent brain, and neuroprotection. Cannabinoid Receptor Interacting Protein 1a (CRIP1a) is associated with neurodevelopment, sensory function, seizures, and mental health (Oliver et al., 2020). CRIP1a is a 10-stranded -barrel protein in the family of lipidated-protein carriers (with UNC119 and PDE6δ) (Booth et al., 2021). Our studies demonstrate that CRIP1a binds myristoylated myrGi N-terminal 9-mer peptide and holo myrGi. Based upon our major advance in knowledge of the structure and function of CRIP1a, we hypothesize that CRIP1a functions to sequester or shuttle Gi proteins, and that the CRIP1a-Gi interaction can be regulated by the CB1R C-terminus during the agonist-stimulated G protein cycle, palmitoylation-depalmitoylation, Arl protein regulatory cycles, and phosphorylation-dephosphorylation to control loading and release of Gi cargo. We propose to investigate the CB1R associated proteins in the N18TG2 and SH-SY5Y human neuroblastoma cell models that endogenously express the CB1R, CRIP1a and G proteins, as well as using in vitro experiments using purified recombinant CRIP1a, myrGi, G and CB1R C-terminal, as well as peptides derived therefrom. The Aims are to determine: 1) selectivity for myrGi as CRIP1a cargo using fluorescence polarization to monitor the binding of peptides from lipidated Gz, Gs, Gq, and G12/13, various lipids (particularly myristate, palmitate), Gi peptide length and amino acid sequence, and to define the mechanism by pulldown assays and X-ray structure determination of these interactions using purified recombinant CRIP1a and peptides or full-length recombinant myrGi, G and CB1R C-terminal; 2) the interface of CRIP1a with the CB1R-G protein activation cycle (mechanism and cellular localization) by quantitating CRIP1a-Gi co-immunoprecipitation from neuronal cell homogenates treated with GTPS, full (2-AG ether, CP55940, WIN55212-2) and partial (methanandamide, Δ9-THC) agonists, competitive antagonist- inverse agonist (SR141716), and after Gi ADP-ribosylation with pertussis toxin. Cellular location of the CRIP1a- Gi interaction will be quantitated using proximity ligation assays and subcellular fractionation; 3) the mechanism(s) for loading and unloading myrGi into CRIP1a by quantitating the gel shifted CRIP1a- Giα by co-immunoprecipitation in intact neuronal cells or homogenates, and determining myrGi-CRIP1a interaction using recombinant proteins to assess: a) roles of palmitoylation and depalmitoylation, b) regulatory properties of Arl proteins, and c) regulation by phosphorylation of predicted sites on the surface of CRIP1a. The results of our investigation of CRIP1a and CB1R at the structural and functional level will inform future investigation of CRIP1a in models of human health and disease.