Cannabinoid Receptor–Interacting Protein 1a Modulates CB₁ Receptor Signaling and Regulation

Abstract

Cannabinoid CB 1 receptors (CB 1 Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP 1a ) binds to the CB 1 R C-terminus and can attenuate constitutive CB 1 R-mediated inhibition of Ca 21 channel activity. We now demonstrate cellular colocalization of CRIP 1a at neuronal elements in the CNS and show that CRIP 1a inhibits both constitutive and agonist-stimulated CB 1 Rmediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP 1a in human embryonic kidney (HEK)-293 cells stably expressing CB 1 Rs (CB 1 -HEK), or in N18TG2 cells endogenously expressing CB 1 Rs, decreased CB 1 Rmediated G-protein activation (measured by agonist-stimulated [ activation. These effects were not attributable to differences in CB 1 R expression or endocannabinoid tone because CB 1 R levels did not differ between cell lines varying in CRIP 1a expression, and endocannabinoid levels were undetectable (CB 1 -HEK) or unchanged (N18TG2) by CRIP 1a overexpression. In CB 1 -HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB 1 Rs and desensitized agonist-stimulated [ 35 S]GTPgS binding. CRIP 1a overexpression attenuated CB 1 R downregulation without altering CB 1 R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP 1a overexpression attenuated both depolarizationinduced 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 CRIP 1a inhibits constitutive CB 1 R activity and demonstrate that CRIP 1a can also inhibit agonist-stimulated CB 1 R signaling and downregulation of CB 1 Rs. Thus, CRIP 1a appears to act as a broad negative regulator of CB 1 R function.

Cannabinoid Receptor Interacting Protein 1a Competition with β-Arrestin for CB1 Receptor Binding Sites

“…CRIP1a overexpression decreased CB 1 Rstimulated [ 35 S]GTPgS binding to Gi3 (Blume et al, 2015), an interaction believed to involve the C-terminus of the CB 1 R (Mukhopadhyay et al, 2000;Mukhopadhyay and Howlett, 2001). Moreover, we found that CRIP1a overexpression in CB 1 R-expressing HEK293 cells inhibited CB 1 R downregulation in response to prolonged agonist occupancy (Smith et al, 2015), whereas genetic deletion of b-arrestin 2 inhibited CB 1 R downregulation in mice (Nguyen et al, 2012). These findings led us to hypothesize that CRIP1a could also influence the interaction of the CB 1 R with b-arrestins, known to function in internalization via the CB 1 R C-terminus (Hsieh et al, 1999;Daigle et al, 2008).…”

“…Cell Culture and Immunocytochemistry Determination of CB 1 R Internalization. We previously reported the development and characterization of stable CRIP1a overexpression (XS-1 and XS-5) and siRNA-knockdown (KD-2C and KD-2F) N18TG2 neuroblastoma clones (passage numbers 30-38) (Blume et al, 2015;Smith et al, 2015). Wild-type, empty-vector control, and transgenic clones were maintained at 37°C under a 5% CO 2 atmosphere in Dulbecco's modified Eagle's medium:Ham's F12 (1:1) complete with GlutaMax, sodium bicarbonate, and pyridoxine-HCl, supplemented with penicillin (100 units/ml) and streptomycin (100 mg/ml) (Gibco Life Technologies, Gaithersburg, MD) and 10% heat-inactivated bovine serum (Gemini Bioproducts, West Sacramento, CA).…”

“…In primary neuronal cortical cultures, it was found that CRIP1a overexpression switched CB 1 R-mediated neuroprotection from an agonist to an antagonist-mediated mechanism (Stauffer et al, 2011). We determined that CRIP1a overexpression in cultured N18TG2 or HEK293 cells was associated with reductions in CB 1 R G-protein-mediated signal transduction (Blume et al, 2015;Smith et al, 2015). CRIP1a overexpression decreased CB 1 Rstimulated [ 35 S]GTPgS binding to Gi3 (Blume et al, 2015), an interaction believed to involve the C-terminus of the CB 1 R (Mukhopadhyay et al, 2000;Mukhopadhyay and Howlett, 2001).…”

In silico interaction analysis of cannabinoid receptor interacting protein 1b (CRIP1b) – CB1 cannabinoid receptor

“…Exogenous CRIP1a reversed CB 1 receptor-mediated tonic inhibition of Ca 2+ currents, whereas CRIP1b could not[11]. CRIP1a-knockdown clones in the model neuroblastoma N18TG2 cells exhibited enhanced ERK1/2 phosphorylation efficacy in response to CP55940 and displayed a leftward shift in CP55940-mediated inhibition of forskolin-stimulated cAMP accumulation [12,13]. CB 1 receptor-mediated G i3 and G o activation by CP55940 was attenuated by CRIP1a over-expression, but robustly enhanced in CRIP1a-knockdown clones.…”

“…Conversely, CP55940-mediated G i1 and G i2 activation was significant enhanced in cells over-expressing CRIP1a, but not inCRIP1a-knockdown clones [14]. These studies suggest that endogenous levels of CRIP1a modulate CB 1 receptor-mediated signal transduction by facilitating a G i/o subtype preference for G i1 and G i2 , accompanied by an overall suppression of G protein-mediated signaling in neuronal cells [12,13]. …”