Discovery and characterization of two novel CB1 receptor splice variants with modified N‐termini in mouse

Abstract

Numerous studies have been carried out in the mouse model, investigating the role of the CB1 cannabinoid receptor. However, mouse CB1 (mCB1) receptor differs from human CB1 (hCB1) receptor in 13 amino acid residues. Two splice variants, hCB1a and hCB1b, diverging in their amino-termini, have been reported to be unique for hCB1 and, via different signaling properties, contribute to CB1 receptor physiology and pathophysiology. We hypothesized that splice variants also exist for the mCB1 receptor and have different signaling properties. On murine hippocampal cDNA, we identified two novel mCB1 receptor splice variants generated by splicing of introns with 117 bp and 186 bp in the N-terminal domain, corresponding to deletions of 39 or 62 amino acids, respectively. The mRNAs for the splice variants mCB1a and mCB1b are expressed at low levels in different brain regions. Western Blot analysis of protein extracts from stably transfected HEK293 cells indicates a strongly reduced glycosylation due to the absence of two glycosylation sites in mCB1b. On-cell Western analysis in these stable lines revealed increased internalization of mCB1a and mCB1b upon stimulation with the agonist WIN55,212-2. Results also point towards an increased affinity to SR141716 for mCB1a, as well as slightly enhanced inhibition of neurotransmission compared to mCB1. In mCB1b, agonist-induced mitogen-activated protein kinase (MAPK) phosphorylation was decreased compared to mCB1 and mCB1a. Identification of mouse CB1 receptor splice variants may help to explain differences found between human and mouse endocannabinoid systems and improve the understanding of CB1 receptor signaling and trafficking in different species.

CB1 Receptor Signaling in the Brain: Extracting Specificity from Ubiquity

“…This advantage may have implications related to the biological functions of the CB 1 receptors ( Onaivi et al , 1999 ). Nevertheless, the presence of splice isoforms both in humans and mice ( Ruehle et al , 2017 ), coming from 5′-UTR introns of the gene, and possible post-translational modifications demonstrates that CB 1 receptors can come in different flavors already at transcriptional and translational level, with potential signaling differences ( Bagher et al , 2013 ; Oddi et al , 2017 ; Straiker et al , 2012 ). Besides these gene expression variables, however, a number of recent observations indicate that CB 1 receptor signaling is pleiotropic and depends on several additional factors, such as its cellular and subcellular localization.…”

Identification of novel mouse and rat CB1R isoforms and in silico modeling of human CB1R for peripheral cannabinoid therapeutics

“…The intra-exonal splicing generates human N-terminal variants of hCB1a 411 with altered and shortened (deletion of 62 amino acids) N termini and hCB1b 439 with a deletion of 33 amino acids from its N terminus [12], in contrast to the alternative splicing of mCB1a 433 and mCB1b 411 , which deletes 39 and 62 amino acids at their respective N termini. The consensus splicing donor sites of mCB1a and mCB1b change from GT to GA and TA, respectively, although splicing acceptor sites remain the consensus AG [35]. In contrast, hCB1a and hCB1b splicing donor sites and acceptor sites agree with the GT-AG consensus sequence [12].…”

“…1a). Together with two additional intra-exon-6 splicing isoforms of mCB1a and mCB1b [35], there were at least five mCB1 isoforms (Fig. 1a).…”

Modulation of Endocannabinoid-Binding Receptors in Human Neuroblastoma Cells by Tunicamycin

“…In particular, hCB 1 b shows a deletion of 33 amino acids that includes Asn 77 and Asn 83, and remarkably this variant has been shown to play a role in metabolic regulation [29]. Recently, two splice variants resembling those of the human receptor were discovered also in the mouse CB 1 -encoding gene CNR1 [30]. Here, the lack of N -glycosylation sites was found to strongly reduce glycosylation level and mitogen-activated protein kinase (MAPK) activity upon CB 1 agonist-induced stimulation [30].…”