PROJECT SUMMARY This new F31 proposal is to support the career development of Erin Hughes who will receive training in cannabinoid receptor biochemical neuropharmacology with Drs. Howlett and Lowther. Recent years have seen a global increase in the usage of marijuana containing the psychoactive constituent Δ9-tetrahydrocannabinol, a partial agonist of the CB1 cannabinoid receptor (CB1R). The CB1R is a G-protein coupled receptor (GPCR) found in the central nervous system and modulates neuroprogenitor development, neural commitment and migration, and neurotransmitter release. GPCR function is dependent upon the ability to change conformations and to transduce a response via G-proteins. This process requires association of a Gα-GDP subunit with Gβγ subunits to form a heterotrimeric G-protein complex that binds to the GPCR. Stimulation of CB1R initiates the exchange of GDP for GTP bound to Gα, activating the G-protein heterotrimer, and releasing the Gα and Gβγ subunits. The released G-protein subunits propagate the signaling cascade through various interactions. Cannabinoid receptor interacting protein 1a (CRIP1a) is a protein found in human neurons at various brain regions, which suppresses CB1R cellular signaling via modulating G-protein activation. The Scientific Premise is that CRIP1a modulation of CB1R can be understood providing us with an in-depth view of the role of CB1R in neuronal cell signaling. CRIP1a can modulate the type of Gα subunit bound to CB1R, however the mechanism of switching is unknown. Previous immunoprecipitation studies demonstrated that the CB1R C-terminal region interacts with CRIP1a, and therefore one possible explanation for CRIP1a-mediated Gα switching may involve formation of a ternary complex between CB1R, subtype-specific Gα proteins, and CRIP1a. The Lowther-Howlett collaboration has determined the high-resolution structure of CRIP1a by X-ray crystallography and found that CRIP1a is a member of the family of carriers for myristoylated or isoprenylated proteins. Based on the knowledge of the structure and function of CRIP1a, I hypothesize that CRIP1a binds N-terminally myristoylated cargo proteins, such as Gαi proteins. I propose to investigate CRIP1a interactions in the N18TG2 neuroblastoma cell model which endogenously expresses the CB1R and associated proteins. The aims of this project are to determine (i) the regulation of the CRIP1a- Gα interaction during agonist stimulation and heterotrimeric G protein dissociation and the cellular localization of such interactions; and (ii) the role of palmitoylation and myristoylation on G-protein interaction with CRIP1a. The results of the proposed studies will prove to be transformative for the field by characterizing a new class of cargo carrying proteins and providing evidence that will advance our understanding of how, when and where CRIP1a interactions modulate CB1R pathways.