Summary The CB1 receptor (CB1R) a therapeutic target for treatment of addictions, neurodegenerative disorders and pain management, but medicinal compounds based upon the CB1R have been limited. The functions of the CB1R to regulate neuronal processes in development, retrograde signaling in neurotransmission, and cellular mechanisms of neuroprotection are critical to brain function. Endocannabinoid ligands 2-arachidonoylglycerol and anandamide are the primary neuromodulators of synaptic activity, but the full understanding of how CB1R signaling can be regulated by associated proteins in specific cell types is just beginning to be appreciated. The Scientific Premise is that CRIP1a modulation of the CB1R can be understood at the structural and functional level such that drug design based on peptide or small molecule interventions can target the CRIP1a-CB1R interaction. Our recently published studies have demonstrated that CRIP1a reduces the density of cell surface CB1R, attenuates the agonist-dependent but not the constitutive internalization processes by competing with ?-arrestins for binding to C-terminal sites, and curtails the trafficking of newly- synthesized CB1R to the cell surface after prolonged WIN55212-2 but not CP55940. Other studies demonstrated that CRIP1a has a critical role in regulating CB1R cellular signaling by altering the preference for coupling from Gi3 & Go, which require the C-terminus for activation, to Gi1 & Gi2, which do not. In unpublished studies, we have determined the high resolution structure from X-ray crystallography, and found that CRIP1a is a member of the family of carriers for myristoylated or isoprenylated proteins. Based upon this major advance in knowledge of the structure and function of CRIP1a, we hypothesize that the function of CRIP1a is to interact with the CB1R?G-protein complex in ways that can be regulated by G-alpha and/or G-gamma subunit specificity, phosphorylation, and interaction with other regulatory proteins that are known to release cargo. We propose to investigate the CB1R associated proteins in the N18TG2 neuroblastoma cell model which endogenously expresses the CB1R and its associated proteins, as well as in in vitro experiments of purified recombinant proteins and peptides derived therefrom. The aims of this project are to investigate: the interaction of CRIP1a with the CB1R; the structural and functional interaction of CRIP1 with G- proteins; and the regulation of CRIP1a function by cargo-releasing proteins and phosphorylation. The results of the proposed investigation should prove to be transformative for the field by providing evidence that CB1R and associated CRIP1a interact to direct cellular signaling pathways. From this understanding, novel peptides and small molecules could be developed as therapeutic agents for neurological diseases in which both CB1R and CRIP1a co-exist in neurons.

R01DA042157

2017-08-15

2022-06-30