JLP, RAA, JOC, BGD, JSM, DES

Cannabinoid CB1 receptors (CB1R) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid Receptor-Interacting Protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca2+ channel activity. We now demonstrate cellular co-localization of CRIP1a at neuronal elements in the CNS, and show that CRIP1a inhibits both constitutive and agoniststimulated CB1R-mediated G-protein activity. Stable over-expression of CRIP1a in HEK- 293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [35S]GTPγS binding) in both cell lines, and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, siRNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not due to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a over-expression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [35S]GTPγS binding. CRIP1a over-expression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation (DSE) and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid, but not adenosine A1 agonists. These results confirm that CRIP1a inhibits This article has not been copyedited and formatted. The final version may differ from this version. Molecular Pharmacology Fast Forward. Published on February 5, 2015 as DOI: 10.1124/mol.114.096495 at ASPET Journals on February 5, 2015 molpharm.aspetjournals.org Downloaded from MOL #96495 5 constitutive CB1R activity, and demonstrate that CRIP1a can also inhibit agoniststimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.