Cannabinoid Receptor Interacting Protein 1a (CRIP1a) Associates with Gαi in a GTPγS or Agonist Dependent Manner

Abstract

<b>Abstract ID 24557</b> <b>Poster Board 568</b> Cannabis usage in recent years has increased due to the legalization of both recreational and medical use, making it important to understand how psychoactive cannabinoids modulate cellular signaling in neurons. The CB1 cannabinoid receptor (CB1R) is a G-protein coupled receptor (GPCR) found in the central nervous system (CNS) and modulates neuroprogenitor development, neural commitment, and neurotransmitter release. Cannabinoid receptor interacting protein 1a (CRIP1a) is an abundant 20 kDa protein in the CNS, which we recently determined to be a beta-barrel protein similar in structure to homologous proteins that serve as intracellular carriers of lipidated proteins. In-vitro binding studies demonstrated that recombinant CRIP1a interacts directly with myristoylated Gαi-derived peptides (Booth et. al. 2021). CRIP1a interacts with the CB1R to modulate its cellular signaling; however, the mechanism by which CRIP1a impacts CB1R-Gi heterotrimer interactions in neurons is unknown. We hypothesized that CRIP1a modulates CB1R signaling via an association with the Gαi subunit after the Gαi subunit is released from the GPCR. We tested this hypothesis by treating N18TG2 mouse neuroblastoma detergent-free cell homogenates with GTPγS to dissociate Gαi. After sedimentation into cytosolic versus membrane fractions, immunoprecipitated CRIP1a co-migrated on SDS-PAGE gels with Gαi with a shift in mobility for these proteins. Immunoblotted CRIP1a and Gαi band densities were quantitated using LI-COR Empiria software for statistical analyses. CRIP1a and Gαi co-migrated as an SDS-resistant complex, consistent with the myristoylated-Gαi intercalating within the hydrophobic interior of the CRIP1a barrel structure. The density of the mobility-shifted complex appeared to move from the membrane to cytosolic fraction within 10 minutes of treatment of the homogenate with GTPγS. The shift from membrane to cytosolic fraction is consistent with Gαi disassociating from the G-protein heterotrimer after binding of the non-hydrolyzable GTP analog. The CB1R full agonist CP55940 was also used to treat N18TG2 cell homogenates within a similar time-course. We further tested the hypothesis by using a proximity ligation assay (PLA) to detect CRIP1a and Gαi within 40 nm of each other, and cellular marker staining in intact N18TG2 cells. N18TG2 cell treatment with 100 nM CP55940 determined that CRIP1a and Gαi co-localize near the plasma membrane (stained with Na/K-ATPase or extracellular CB1R antibodies) within 30-90 seconds of agonist treatment. Collectively, these data suggest CRIP1a is a cargo-carrying protein capable of binding the myristoylated-Gαi subunit near the plasma membrane in a GTPγS and agonist-dependent manner. These studies were supported by NIH grants R01-DA042157, and F31-DA056188, and the Wake Forest University Cowgill Fellowship.