Figure 1
Human CNR1 Gene Splice Variants.
Human CNR1 (6q15) gene structure (A) and its alternatively spliced transcript variants (B): the open bar represents the exons with exon numbers inside and horizontal lines represent the introns. Human CNR1 has splicing sites in two of its exons (A). Within exon 1 there are two splicing sites (1A and 1B) and within coding exon 4 there are four splicing sites (4A-D) indicated by arrows above the exons. Alternatively-spliced transcript variants are shown (B) and GenBank accession numbers are included in parenthesis; the corresponding protein isoforms are indicated with an arrow. CB1 receptor full length protein and N-terminal isoforms are represented by CB1, CB1a and CB1b, respectively. CB1 full length amplicons align the common deleted region in between exon 4C-4D, recognizing all full length splice variants. Each TaqMan probe recognizes the adjacent and inter-exonal spliced exons and is represented by short horizontal bars in black, grey and white for CB1, CB1a and CB1b receptors, respectively. The specific full length CB1 receptor amplicon (black bar) aligns the intra-exonal sequence between splicing sites 4B and 4C, and does not recognize the spliced variants CB1a and CB1b (C). Alignment of the protein N-terminal amino acid sequences of CB1a and CB1b with full length CB1 receptor (D). Gaps are represented by dashed lines, identical amino acids by the asterisks. The beginning of the first transmembrane domain is underlined. Increasing concentrations of ACEA did not influence on Ex4-mediated cAMP accumulation in CHO cells when the cells were not transduced with any CB1 receptor (E). Cells were pre-treated with ACEA for 20 min before addition of Ex4 for a further 15 min. **p < 0.01, n.s. = not significant compared to vehicle. Treatment with the synthetic CB1 receptor agonist ACEA attenuated Ex4-mediated cAMP accumulation in CHO-GLP-1R cells when transduced with CB1 and CB1a, and to a greater extent with CB1b (C). All values were normalized to protein concentration. Data represent mean percentage over maximum response ± SEM from at least three independent experiments. ***p ≤ 0.005 compared to CB1 full length. For cell line validation, see Supplementary Fig. S1.
