Cloning delta receptors

Distinct genes for delta opioid receptor subtypes however, have not been identified, reducing the probability that delta opioid receptor subtypes exist as distinct molecular entities. On the other hand, this does not eliminate the possibility that pharmacological delta opioid receptor subtypes can be produced by alternative splicing from the cloned delta receptor genomic sequence, as was found for the other opioid receptors [28]. However, there is no evidence for alternative splicing for the delta opioid receptor of this at... 

Tsu R, Chan J, Wong Y. Regulation of multiple effectors by the cloned delta opioid receptor stimulaton of phospholi-... 

Prodynorphin contains three copies of Leu-enkephalin with carboxy-termi-nus extended polypeptides of various lengths known as dynorphin A (or dynorphin 1-17), dynorphin B (dynorphin 1-13), or a- and 3-neoendorphin. These peptides derived from prodynorphin are selective to kappa receptors and can also be further broken down to Leu-enkephalin. The identification of the delta receptor (or the enkephalin receptor) was a direct consequence of the discovery of enkephalins. This chapter will review the major events that are important for the identification of delta receptors and the subsequent cloning of delta receptor genes, and eventually all other opioid receptor genes. 

Cloning of the opioid receptors did not come easy, and there were many false claims along the way. Efforts began in the mid-1980s in the wake of cloning of the opioid peptide precursors. However, it was not till 1992 that the delta receptor was first cloned and provided the critical probes leading to the characterization of the entire family of opioid receptors. 

The cloning of the delta receptor set in motion a competitive race to identify other members of the opioid receptor family. Homologous orphan clones were quickly assessed for opioid receptor binding properties, which resulted in the identification of the kappa receptor and reassignment of an orphan clone as the delta opioid receptor [31]. PCR, genomic, and cDNA screens revealed the mu opioid receptor and an extremely abundant orphan member, named opioid receptor-like (ORL-1) receptor (reviewed by Massotte and Kieffer [30]). 

Rothman and Westfall [3,4], divided the delta receptor population into two components one coupled to mu opioid receptors, and the other acting alone. The third hypothesis combined these ideas, by suggesting that the delta receptors that interact with the mu opioid receptors are different molecular entities from those that act alone [5,6]. A definitive test of the above hypotheses would be to identify two or more delta receptor proteins by molecular cloning. The cloning of the human delta opioid receptor [7] was the fortunate outcome of a larger project in our laboratory to clone of the cDNAs encoding putative delta opioid receptor subtypes. 

Both groups used expression cloning to screen cDNA libraries obtained from NG 108-15 mouse neuroblastoma-rat glioma hybridoma cells. These cells were a logical choice for delta opioid receptor cloning efforts, since they express high density of delta receptors and can be produced in great quantities in cell culture. 

Since at the time of the described studies it was thought that the two delta receptor antagonists naltriben (NTB) and 7-benzylidenenaltrexone (BNTX) were selective for the proposed delta-2 and delta-1 receptor subtypes, respectively [19], the relatively high affinity of NTB for the expressed receptors (as compared to BNTX, see Table 1) was thought to be consistent with the cloned human delta receptor being of the delta-2 opioid receptor subtype. 

The delta receptor was the first opioid receptor to be cloned in 1992, almost 20 years after opioid binding sites were discovered in the brain [1,2]. Cloning was achieved simultaneously by two independent teams, using an expression strategy [3,4]. Homology cloning techniques then delivered the whole opioid... 

In vitro and in vivo studies conducted in the 1980s and 1990s clearly demonstrated synergistic interactions in vivo between mu and delta receptors that were most simply explained by a mu-delta opioid receptor complex. The failure to clone opioid receptors corresponding to the postulated mu-delta opioid receptor complex, as well as other opioid receptor subtypes defined... 

Subsequent to the molecular cloning and cDNA sequence analysis of the delta opioid receptor, antireceptor antibodies were raised against synthetic peptides based on the deduced amino acid sequence of this receptor. To date, there have been a limited number of studies using antibodies directed toward the amino terminus of the cloned murine receptor to assess the distribution of delta opioid receptorlike immunoreactivity in the gastrointestinal tract, and these have been confined so far to the porcine small intestine. They have shown the presence of delta opioid receptor-immuno-reactive neurons and fibers in both the myenteric and submucosal plexuses and as well as in myenteric neurons maintained in primary culture [22, 23]. Receptor-like immunoreactivity in neuronal cell bodies appears to be localized in the cytoplasm and is likely to be trafficked to nerve terminals. These neurons are coimmunoreactive for the acetylcholine-synthesizing... 

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