Dataset Information

Molecular Interaction between Distal C-Terminal Domain of the CB1 Cannabinoid Receptor and Cannabinoid Receptor Interacting Proteins (CRIP1a/CRIP1b).

ABSTRACT: We have investigated the structure of the distal C-terminal domain of the of the CB1 cannabinoid receptor (CB1R) to study its interactions with CRIP1a and CRIP1b using computational techniques. The amino acid sequence from the distal C-terminal domain of CB1R (G417-L472) was found to be unique, as it does not share sequence similarity with other protein structures, so the structure was predicted using ab initio modeling. The computed model of the distal C-terminal region of CB1R has a helical region between positions 441 and 455. The CRIP1a and CRIP1b were modeled using Rho-GDI 2 as a template. The three-dimensional model of the distal C-terminal domain of the CB1R was docked with both CRIP1a as well as CRIP1b to study the crucial interactions between CB1R and CRIP1a/b. The last nine residues of CB1R (S464TDTSAEAL4722) are known to be a CRIP1a/b binding site. The majority of the key interactions were identified in this region, but notable interactions were also observed beyond theses nine residues. The multiple interactions between Thr418 (CB1R) and Asn61 (CRIP1a) as well as Asp430 (CB1R) and Lys76 (CRIP1a) indicate their importance in the CB1R-CRIP1a interaction. In the case of CRIP1b, multiple hydrogen bond interactions between Asn437 (CB1R) and Glu77 (CRIP1b) were observed. These interactions can be critical for CB1R's interaction with CRIP1a/b, and targeting them for further experimental studies can advance information about CRIP1a/b functionality.

SUBMITTER: Singh P

PROVIDER: S-EPMC7271255 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Molecular Interaction between Distal C-Terminal Domain of the CB1 Cannabinoid Receptor and Cannabinoid Receptor Interacting Proteins (CRIP1a/CRIP1b).

Singh Pratishtha P Ganjiwale Anjali A Howlett Allyn C AC Cowsik Sudha M SM

Journal of chemical information and modeling 20191210 12

We have investigated the structure of the distal C-terminal domain of the of the CB1 cannabinoid receptor (CB1R) to study its interactions with CRIP1a and CRIP1b using computational techniques. The amino acid sequence from the distal C-terminal domain of CB1R (G417-L472) was found to be unique, as it does not share sequence similarity with other protein structures, so the structure was predicted using ab initio modeling. The computed model of the distal C- ...[more]

PMID: 31769975

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Project description:CB1 cannabinoid receptors (CB1R) are one of the most abundantly expressed G protein coupled receptors (GPCR) in the CNS and regulate diverse neuronal functions. The identification of GPCR interacting proteins has provided additional insight into the fine-tuning and regulation of numerous GPCRs. The cannabinoid receptor interacting protein 1a (CRIP1a) binds to the distal carboxy terminus of CB1R, and has been shown to alter CB1R-mediated neuronal function [1]. The mechanisms by which CRIP1a regulates CB1R activity have not yet been identified; therefore the focus of this investigation is to examine the cellular effects of CRIP1a on CB1R signaling using neuronal N18TG2 cells stably transfected with CRIP1a over-expressing and CRIP1a knockdown constructs. Modulation of endogenous CRIP1a expression did not alter total levels of CB1R, ERK, or forskolin-activated adenylyl cyclase activity. When compared to WT cells, CRIP1a over-expression reduced basal phosphoERK levels, whereas depletion of CRIP1a augmented basal phosphoERK levels. Stimulation of phosphoERK by the CB1R agonists WIN55212-2, CP55940 or methanandamide was unaltered in CRIP1a over-expressing clones compared with WT. However, CRIP1a knockdown clones exhibited enhanced ERK phosphorylation efficacy in response to CP55940. In addition, CRIP1a knockdown clones displayed a leftward shift in CP55940-mediated inhibition of forskolin-stimulated cAMP accumulation. CB1R-mediated Gi3 and Go activation by CP99540 was attenuated by CRIP1a over-expression, but robustly enhanced in cells depleted of CRIP1a. Conversely, CP55940-mediated Gi1 and Gi2 activation was significant enhanced in cells over-expressing CRIP1a, but not in cells deficient of CRIP1a. These studies suggest a mechanism by which endogenous levels of CRIP1a modulate CB1R-mediated signal transduction by facilitating a Gi/o protein subtype preference for Gi1 and Gi2, accompanied by an overall suppression of G-protein-mediated signaling in neuronal cells.

Project description: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 Ca(2+) 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 [(35)S]GTP?S (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 [(35)S]GTP?S 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.

Dataset Information

Molecular Interaction between Distal C-Terminal Domain of the CB1 Cannabinoid Receptor and Cannabinoid Receptor Interacting Proteins (CRIP1a/CRIP1b).

Publications

Molecular Interaction between Distal C-Terminal Domain of the CB<sub>1</sub> Cannabinoid Receptor and Cannabinoid Receptor Interacting Proteins (CRIP1a/CRIP1b).

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