Abstract
Cannabinoid CB1 receptors (CB1Rs) 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 colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide–binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (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 (guanylyl-5′-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA–mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable 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 overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [35S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.
Footnotes
- Received October 21, 2014.
- Accepted January 26, 2015.
This work was supported by the National Institutes of Health National Institute on Drug Abuse [R21-DA025321, R01-DA003690, R03-DA035424, F31-DA023747, F31-DA032215, R01-DA011322, K05-DA021696, and T32-DA007027], the National Institute on Neurologic Disorders and Stroke [T32-NS007288], the National Eye Institute [R21-EY021831], the Biotechnology and Biological Sciences Research Council [S19916], and the National Center for Advancing Translational Sciences [UL1-TR000058] in support of the A.D. Williams Fund of Virginia Commonwealth University. Mass spectrometry was supported in part with funding from the National Institute on Drug Abuse [P30-DA033934]. Rat microscopy was performed at the VCU Department of Anatomy and Neurobiology Microscopy Facility, supported in part with funding from the National Institute on Neurologic Disorders and Stroke [P30-NS047463].
↵This article has supplemental material available at molpharm.aspetjournals.org.
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics
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