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

Numerous studies have been carried out in the mouse model, investigating the role of the cannabinoid receptor type 1 (CB1). 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 because of 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 as compared to mCB1. Results also point toward an increased affinity to SR141716 for mCB1a, as well as slightly enhanced inhibition of neurotransmission compared to mCB1. In mCB1b, agonist-induced 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.