Cannabinoid receptor interacting protein (CRIP1a) attenuates CB1R signaling in neuronal cells

Blume, Lawrence C.; Eldeeb, Khalil; Bass, Caroline E.; Selley, Dana E.; Howlett, Allyn C.

doi:10.1016/j.cellsig.2014.11.006

Cited by 35 publications

(58 citation statements)

References 35 publications

(58 reference statements)

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“…[ 35 S] GTPγS binding to activated Gαi/o proteins was performed on membranes prepared from the control and RGS2 knockdown cells using the procedure previously described (Blume et al, 2015). Briefly, cells were homogenized in cold hypotonic buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol and the Protease Inhibitor Cocktail) followed by centrifugation at 1,000 × g for 10 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

et al. 2016

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Dysregulated expression and function of dopamine D2 receptors (D2Rs) are implicated in drug addiction, Parkinson’s disease and schizophrenia. In the current study, we examined whether D2Rs are modulated by regulator of G protein signaling 2 (RGS2), a member of the RGS family that regulates G protein signaling via acceleration of GTPase activity. Using neuroblastoma 2a (N2A) cells, we found that RGS2 was immunoprecipitated by aluminum fluoride-activated Gαi2 proteins. RGS2 siRNA knockdown enhanced membrane [35S] GTPγS binding to activated Gαi/o proteins, augmented inhibition of cAMP accumulation and increased ERK phosphorylation in the presence of a D2/D3R agonist quinpirole when compared to scrambled siRNA treatment. These data suggest that RGS2 is a negative modulator of D2R-mediated Gαi/o signaling. Moreover, RGS2 knockdown slightly increased constitutive D2R internalization and markedly abolished quinpirole-induced D2R internalization assessed by immunocytochemistry. RGS2 knockdown did not compromise agonist-induced β-arrestin membrane recruitment; however, it prevents β-arrestin dissociation from the membrane after prolonged quinpirole treatment during which time β-arrestin moved away from the membrane in control cells. Additionally, confocal microscopy analysis of β-arrestin post-endocytic fate revealed that quinpirole treatment caused β-arrestin to translocate to the early and the recycling endosome in a time-dependent manner in control cells whereas translocation of β-arrestin to these endosomes did not occur in RGS2 knockdown cells. The impaired β-arrestin translocation likely contributed to the abolishment of quinpirole-stimulated D2R internalization in RGS2 knockdown cells.

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Section: Methodsmentioning

confidence: 99%

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

et al. 2016

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“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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]. …”

Section: Introductionmentioning

confidence: 99%

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

Singh

Ganjiwale

Howlett

et al. 2017

Journal of Molecular Graphics and Modelling

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Cannabinoid Receptor Interacting Protein isoform 1b (CRIP1b) is known to interact with the CB1 receptor. Alternative splicing of the CNRIP1 gene produces CRIP1a and CRIP1b with a difference in the third exon only. Exons 1 and 2 encode for a functional domain in both proteins. CRIP1a is involved in regulating CB1 receptor internalization, but the function of CRIP1b is not very well characterized. Since there are significant identities in functional domains of these proteins, CRIP1b is a potential target for drug discovery. We report here predicted structure of CRIP1b followed by its interaction analysis with CB1 receptor by in-silico methods. A number of complementary computational techniques, including, homology modeling, ab-initio and protein threading, were applied to generate three-dimensional molecular models for CRIP1b. The computed model of CRIP1b was refined, followed by docking with C terminus of CB1 receptor to generate a model for the CRIP1b- CB1 receptor interaction. The structure of CRIP1b obtained by homology modelling using RHO_GDI-2 as template is a sandwich fold structure having beta sheets connected by loops, similar to predicted CRIP1a structure. The best scoring refined model of CRIP1b in complex with the CB1 receptor C terminus peptide showed favourable polar interactions. The overall binding pocket of CRIP1b was found to be overlapping to that of CRIP1a. The Arg82 and Cys126 of CRIP1b are involved in the majority of hydrogen bond interactions with the CB1 receptor and are possible key residues required for interactions between the CB1 receptor and CRIP1b.

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“…A recently published article by some of the coauthors of this paper showed that manipulation of CRIP 1a expression in N18TG2 cells modulated constitutive and agonist-stimulated extracellular signal-regulated kinase 1/2 phosphorylation by the CB 1 R, and that CB 1 R coupling to Ga o and Ga i 3 was attenuated, whereas coupling to Ga i 1 and 2 was enhanced, by overexpression of CRIP 1a (Blume et al, 2015).…”

Section: Note Added In Proofmentioning

confidence: 99%

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

et al. 2015

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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.

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Cannabinoid receptor interacting protein (CRIP1a) attenuates CB1R signaling in neuronal cells

Cited by 35 publications

References 35 publications

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

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

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

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Cannabinoid receptor interacting protein (CRIP1a) attenuates CB1R signaling in neuronal cells

Cited by 35 publications

References 35 publications

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

RGS2 modulates the activity and internalization of dopamine D2 receptors in neuroblastoma N2A cells

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

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

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