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Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.

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 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.

SUBMITTER: Smith TH

PROVIDER: S-EPMC4366794 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.

Smith Tricia H TH Blume Lawrence C LC Straiker Alex A Cox Jordan O JO David Bethany G BG McVoy Julie R Secor JR Sayers Katherine W KW Poklis Justin L JL Abdullah Rehab A RA Egertová Michaela M Chen Ching-Kang CK Mackie Ken K Elphick Maurice R MR Howlett Allyn C AC Selley Dana E DE

Molecular pharmacology 20150205 4

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 C ...[more]

PMID: 25657338

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Project description:Background: The endocannabinoid system is present in multiple organ systems and is involved in smooth muscle regulation, immune function, neuroendocrine modulation, and metabolism of tissues. Limited data are available regarding the presence and role of this system in reproductive tissues. Components of the endocannabinoid system have been identified in myometrial and placental tissues. However, no study has investigated differential expression of the endocannabinoid system in labor. Objectives: The purpose of this study was to identify and quantify two components of the endocannabinoid system, the CB1 cannabinoid receptor and cannabinoid receptor interacting protein 1a (CRIP1a) in uterine and placental tissues, and to determine if there is differential expression in tissues exposed to labor. We hypothesized that CB1 cannabinoid receptor concentration would be altered in uterine and placental tissue exposed to labor compared with tissues not exposed to labor. Study Design: Uterine and placental tissue samples were collected in nine laboring and 11 nonlaboring women undergoing cesarean delivery. CB1 cannabinoid receptor and CRIP1a presence and quantification were evaluated using western blot, immunohistochemistry, and real-time quantitative polymerase chain reaction. Statistical comparisons of laboring and nonlaboring subjects were made for uterine and placental tissue using a Mann-Whitney test. Results: Immunohistochemistry demonstrated positive staining for CB1 cannabinoid receptors and CRIP1a in uterine tissue. The protein abundance of CB1 cannabinoid receptor in uterine tissue was significantly lower in tissues exposed to labor (p=0.01). The protein abundance of CRIP1a was lower in uterine tissue exposed to labor but did not reach statistical significance (p=0.06). mRNA expression of CB1 cannabinoid receptor (p=0.20) and CRIP1a (p=0.63) did not differ in labored compared with nonlabored uterine tissues. Conclusions: Our findings of diminished protein density of CB1 cannabinoid receptor in uterine tissue exposed to labor support the hypothesis that the endocannabinoid system plays a role in parturition. Our data add to the growing body of evidence indicating the endocannabinoid system is of importance for successful reproduction and support the need for additional research investigating this complex system as it pertains to labor. ClinicalTrials.gov ID: NCT03752021.

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.

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Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.

Publications

Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.

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