Cannabinoid receptor interacting protein (CRIP1a) attenuates CB1R signaling in neuronal cells
Introduction
The CB1 cannabinoid receptor (CB1R) belongs to the class A rhodopsin-like G protein coupled receptor (GPCR) family. CB1Rs display highest expression in the nervous systems [2], [3], [4], where they have been implicated in numerous physiological processes, including but not limited to energy balance, neuroprotection, pain, and cellular differentiation and proliferation [5]. Based on the location and function of CB1Rs in the CNS, it is no surprise that CB1Rs provide a potentially promising therapeutic target for a diverse number of diseases and disorders [6]; however, the clinical utility and success of CB1R therapeutic agents has been impeded as a result of untoward side-effect profiles.
CB1R signaling is mediated by pertussis toxin-sensitive Gi/o proteins, and leads to inhibition of adenylyl cyclase (AC), regulation of ion channels, induction of immediate early gene expression, and activation of members of the mitogen-activated protein kinase (MAPK) family including extra-cellular regulated kinase 1/2 (ERK1/2) [7]. Studies using peptides mimicking specific regions of CB1R's C-terminus or intracellular loop 3 have demonstrated a preference in binding of specific G proteins to different regions of CB1R. Gαi1 and Gαi2 have been reported to interact with the third intracellular loop of CB1R [8], [9], whereas Gαi3 and Gαo primarily interact with the juxtamembrane C-tail domain [8] of CB1R. Additionally, specificity in G protein activation appears to occur in a ligand-dependent manner [10], suggesting that upon binding, CB1R ligands can induce differences in receptor conformational changes, which can lead to the coupling and activation of specific G protein subtypes.
The GPCR C-terminal tail is a major site for protein-protein interactions, and although G protein binding is a key component in GPCR signaling, it is now well appreciated that other modulatory proteins are involved in receptor activity-dependent and G protein selective signaling [11], [12]. The cannabinoid receptor interacting protein (CRIP1a), which binds to the distal C-terminal tail of CB1R, was initially characterized for its ability to reverse CB1R-mediated tonic inhibition of Ca2 + channels in superior cervical ganglion neurons [1]. Studies using a cell culture model of glutamate neurotoxicity in primary neuronal cortical neurons, showed that lentiviral over-expression of CRIP1a reversed CB1R-mediated neuroprotection from an agonist- to antagonist-driven mechanism [13]. However, the underlying mechanism responsible for this modification of CB1R ligand-mediated neuroprotection is unknown.
To explore the emerging roles of CRIP1a in regulating CB1R, our laboratory developed CRIP1a gain and loss of function transgenic neuronal clones, and reported preliminary observations of alterations in agonist-promoted CB1R activation and internalization by CRIP1a [14], [15]. In the present study, we determined the effect of CRIP1a on CB1R signaling and the associated downstream consequences on cellular function. Both CRIP1a over-expression and RNA interference-induced CRIP1a knockdown were examined in stably-transfected clones of the N18TG2 neuronal cell line, which endogenously expresses both CB1R and CRIP1a. The focus was on CB1R- Gαi/o-mediated inhibition of cAMP production and Gβγ-mediated MAPK activation. Herein we demonstrate that CRIP1a functions as a negative modulator of CB1R cellular signaling, as depletion of CRIP1a increased the interaction with Gi3 and Go subtypes, increased the potency of CB1R agonists to inhibit forskolin-stimulated cAMP accumulation, and enhanced the efficacy of CB1R agonist-stimulated ERK phosphorylation. These studies suggest a role for CRIP1a in CB1R signaling and modulation of agonist-mediated G protein coupling.
Section snippets
Cell culture and generation of stable neuronal CRIP1a transgenic clones
N18TG2 neuroblastoma cells and stable clones were cultured and maintained in complete media containing Dulbecco's Modified Eagle's Medium (DMEM):Ham's F-12 (1:1) with GlutaMax, sodium bicarbonate, and pyridoxine-HCl, supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml) (Gibco Life Technologies, Gaithersburg, MD, USA) and 10% heat-inactivated bovine serum (JRH Biosciences, Lenexa, KS, USA), and incubated at 37 °C in a humidified atmosphere containing 95% air and 5% carbon
CRIP1a influences CB1R cell surface equilibrium but not mRNA or total protein levels
In order to better understand the cellular mechanisms involved in the regulation of CB1R by CRIP1a, we developed stable neuronal transgenic CRIP1a over-expressing (XS) and knockdown (KD) clones in the N18TG2 cell line. Based on their CRIP1a mRNA and protein expression, two different CRIP1a XS and KD clones were selected for the present investigation. CRIP1a XS clones 1 and 5 express CRIP1a:CB1R mRNA levels that are 12:1 (XS 1) and 7:1 (XS 5), compared with a 1:7 ratio in WT cells (Fig. 1A).
Discussion
In addition to direct stimulation of the receptor by agonists, signaling by CB1R can be further modified by accessory proteins, such as β-arrestin, G protein Associated Sorting Proteins (GASP), and CRIP1a [11], [12], [15]. CRIP1a was initially characterized for its ability to bind to a segment of the distal C-terminal of the CB1R [1]. Following that report, studies of retinal circuitry showed that CRIP1a could be found in amacrine cells, and in certain cone (but not rod) terminals [33]. The CB1
Conclusions
The knowledge gained regarding the effects of CRIP1a on CB1R signaling demonstrates how CB1R function and activity can be fine-tuned by accessory proteins. We determined that reduction in CRIP1a protein levels increased CB1R agonist-stimulated Gi3/o protein activation and promoted inhibition of forskolin-stimulated cAMP accumulation in neuroblastoma cells. Depletion of CRIP1a enhanced CB1R-mediated maximal ERK phosphorylation, a process that was abolished by blocking CB1R internalization. This
Acknowledgements
This work was supported by US Public Health Services grants: R01-DA03690 (ACH), R21-DA025321 (ACH and DES), K01-DA024763 (CEB), P50-DA006634 (ACH, CEB, KE), K12-GM102773 (KE), T32-DA00724 and F31-DA032215 (LCB). The authors declare no conflicts of interest.
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These authors contributed equally to this work.