Endocannabinoid signaling depends upon the CB₁ and CB₂ cannabinoid receptors, their endogenous ligands anandamide and 2-arachidonoylglycerol, and intracellular proteins that mediate responses via the C-terminal and other intracellular receptor domains. The CB₁ receptor regulates and is regulated by associated G proteins predominantly of the Gi/o subtypes, β-arrestins 1 and 2, and the cannabinoid receptor-interacting protein 1a (CRIP1a). Evidence for a physiological role for CRIP1a is emerging as data regarding the cellular localization and function of CRIP1a are generated. Here we summarize the neuronal distribution and role of CRIP1a in endocannabinoid signaling, as well as discuss investigations linking CRIP1a to development, vision and hearing sensory systems, hippocampus and seizure regulation, and psychiatric disorders including schizophrenia. We also examine the genetic and epigenetic association of CRIP1a within a variety of cancer subtypes. This review provides evidence upon which to base future investigations on the function of CRIP1a in health and disease.

Cannabinoid receptor interacting protein 1a (CRIP1a) modulates CB₁ cannabinoid receptor G-protein coupling in part by altering the selectivity for Gα_i subtype activation, but the molecular basis for this function of CRIP1a is not known. We report herein the first structure of CRIP1a at a resolution of 1.55 Å. CRIP1a exhibits a 10-stranded and antiparallel β-barrel with an interior comprised of conserved hydrophobic residues and loops at the bottom and a short helical cap at the top to exclude solvent. The β-barrel has a gap between strands β8 and β10, which deviates from β-sandwich fatty acid-binding proteins that carry endocannabinoid compounds and the Rho-guanine nucleotide dissociation inhibitor predicted by computational threading algorithms. The structural homology search program DALI identified CRIP1a as homologous to a family of lipidated-protein carriers that includes phosphodiesterase 6 delta subunit and Unc119. Comparison with these proteins suggests that CRIP1a may carry two possible types of cargo: either (i) like phosphodiesterase 6 delta subunit, cargo with a farnesyl moiety that enters from the top of the β-barrel to occupy the hydrophobic interior or (ii) like Unc119, cargo with a palmitoyl or a myristoyl moiety that enters from the side where the missing β-strand creates an opening to the hydrophobic pocket. Fluorescence polarization analysis demonstrated CRIP1a binding of an N-terminally myristoylated 9-mer peptide mimicking the Gα_i N terminus. However, CRIP1a could not bind the nonmyristolyated Gα_i peptide or cargo of homologs. Thus, binding of CRIP1a to Gαi proteins represents a novel mechanism to regulate cell signaling initiated by the CB₁ receptor.

ID 24557 Poster Board 568 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.