The mouse OPRM1
gene undergoes extensive splicing to generate multiple mu subtypes, confirming their initial suggestion (Wolozin and Pasternak 1981
). The functional relevance of the mouse splice variants has been suggested by anti-sense and KO models, including recent studies with the exon 11 KO mice. Disruption of MOR-1 exon 1 eliminated morphine actions, but not those of heroin and morphine-6β-glucurnoide (Schuller et al. 1999
). This residual analgesia was not because of cross activation of delta and kappa1
receptors since it was retained in a mu/delta/kappa1
triple KO mouse (M. King, J. Pintar and G.W. Pasternak, in preparation), suggesting a possible role for exon 11-associated variants that were still expressed in these mice (unpublished observation). On the other hand, elimination of the exon 11-associated variants had little effect on morphine and methadone analgesia, but significantly impaired the analgesic activity of heroin and M6G (Y.X. Pan, J. Xu, M. Xu, G. Rossi, J. Matulonis and G.W. Pasternak, submitted), strongly implying a role for exon 11-associated variants in heroin, and not morphine, actions. The dissociation of morphine and heroin actions in the mouse may prove important in understanding both opioid analgesia and addiction.
The primary question in the current study was whether or not a similar 5′ splicing profile existed in humans. Thus, our isolation of a human homolog of the mouse exon 11 along and three human exon 11-associated splice variants in both brain and a human neuroblastoma cell line raises the possibility that a similar pharmacological dissociation between morphine and heroin exists in people. Our findings, in conjunction with additional studies identifying seven homologous exon 11-associated variants in the rat (J. Xu, M. Xu, G.W. Pasternak and Y.X. Pan, in preparation), suggests conservation of exon 11 and its associated splice variants across species. With the new exon 11-associated variants, the human OPRM1 gene now contains at least 10 exons spanning over 200 kb, a size comparable to ~250 kb of the mouse OPRM1 gene that generates at least 17 splice variants.
Overall, splicing of the human OPRM1
gene corresponds closely with that in the mouse. The exon 11 homologs are located ~30 kb upstream of exon 1 in the mouse and ~28 kb in humans. However, the human exon 11 is longer than mouse exon 11 and contains an alternative splice site that divides the human exon 11 into two parts, a splicing pattern not seen present in the mouse exon 11. The mouse contains a separate promoter located just upstream from exon 11 that controls the expression of the nine exon 11-associated splice variants (Pan 2002
; Xu et al. 2006
). The location of the human exon 11 approximately 28 kb upstream from exon 1 raises the obvious question of whether a similar human exon 11 promoter exists at upstream of exon 11. Preliminary studies have suggested that the 5′
flanking region of the human exon 11 contains promoter activity, as demonstrated in human neuroblastoma cell lines using a secreted alkaline phosphotase reporter assay (J. Xu, M. Xu and X.Y. Pan, in preparation).
The mouse has three exon 11-associated variants that contain exon 1 and predict full length receptor proteins identical to that of mMOR-1 itself. In humans, hMOR-1i contains exon 1 and predicts a full length receptor very similar to hMOR-1. The difference at the protein level between hMOR-1i and hMOR-1 is because of the presence of an additional initial 93 amino acids at the tip of the N-terminus. hMOR-1i was the first splice variant isolated so far from OPRM1 genes that predicts a receptor with additional amino acid sequences extended at the N-terminus of hMOR-1. It was not surprising that the pharmacological binding profiles of hMOR-1i were indistinguishable with those of hMOR-1 since their predicted binding pockets involve identical transmembrane domains. However, differences in agonist-induced G protein coupling assessed with [35S]GTPγS binding assay raises a number of interesting questions. Foremost is how the additional extracellular 93 amino acids at the tip of the N-terminus can alter receptor-G-protein coupling which is presumably more dependent upon the intracellular structures, as well as how can these changes influence transduction without changing opioid binding.
A number of exon 11-associated variants in mice predict truncated variants. Several predict very small (< 10 kDa) proteins that can be generated with in vitro
translation, but that have not yet been isolated from brain. However, the 6 TM variants mMOR-1G, mMOR-1M and mMOR-1N are expressed in mouse brain (Pan et al. 2001
). Although the functional significance of these truncated variants remains unclear, evidence is mounting for their relevance. [3
H]Diprenorphine can label the mouse 6 TM variants (KD
~10 nM) and the mouse 6 TM variants are able to modulate the expression and function of mMOR-1 when they were co-expressed (Y.X. Pan, J. Xu and G.W. Pasternak, unpublished observation). Furthermore, they can physically associate with, and modulate the activity of, traditional full length MOR-1 variants (X.Y. Pan, J. Xu, M. Xu and G.W. Pasternak, unpublished observations). The functional significance of the mouse exon 11-associated splice variants was further supported by finding in an exon 11 KO mouse. Disrupting exon 11 greatly diminished the analgesic actions of M6G, fentanyl and heroin, while the analgesic activity of morphine and methadone were not affected. Similar 6 TM variants have now been isolated from humans. Like the mouse 6 TM variants, both hMOR-1G1 and hMOR-1G2 produced a 6 TM protein, as illustrated by in vitro
translation studies. The conservation of these variants in mice, rats and humans is consistent with a functional significance.
The 6 TM variants lack the first TM encoded by exon 1. This is interesting since there is another human variant comprised only of TM 1 (Du et al. 1997
; Pan 2005
), the one missing in these new human variants. Preliminary studies with the mouse variants indicate that the single TM and the 6 TM variants can physically associate, raising the question of whether the complexed variants might complement each other to form a complete 7 TM receptor (X.Y. Pan and G.W. Pasternak, unpublished observations). However, the functional effects of these dimers have not yet been documented.
Finally, the regional distribution of the exon 11-associated variants varied markedly among brain regions, implying cell-specific and region-specific mRNA processing. This is similar to the mouse, which also demonstrated differences in their distribution at both the mRNA and protein level (Pan et al. 2001
; Abbadie et al. 2004
In conclusion, the discovery of human exon 11 and its associated splice variants further illustrates the complexity of the human OPRM1 gene and the overall conservation of the splicing patterns among mice, rats and humans. The importance of the exon 11-associated variants in mice in mediating the actions of heroin and M6G suggests that these human exon 11-associated variants may also prove important clinically and may help explain the diverse responses of patients to the various mu opioids.