Cloning of opioid receptors

The opioid peptides vary in their binding affinities for the multiple opioid receptor types. Leu- and Met-enkephalin have a higher affinity for 5-receptors than for the other opioid receptor types (68), whereas the dynorphin peptides have a higher affinity for K-sites (69). P-Endorphin binds with equal affinity to both p- and 5-receptors, but binds with lower affinity to K-sites (70). The existence of a P-endorphin-selective receptor, the S-receptor, has been postulated whether this site is actually a separate P-endorphin-selective receptor or is a subtype of a classical opioid receptor is a matter of controversy (71,72). The existence of opioid receptor subtypes in general is quite controversial although there is some evidence for subtypes of p- (73), 5-(74), and K-receptors (72,75), confirmation of which may be obtained by future molecular cloning studies. 

Opioids act on heptahelical G-protein-coupled receptors. Three types of opioid receptors (p, 8, k) have been cloned. Additional subtypes (e.g., pl3 p2, 81 82), possibly resulting from gene polymorphisms, splice variants or alternative processing have been proposed. Opioid receptors are localized and can be activated... 

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

Kong H, Raynor K, Yano H et al. Agonists and antagonists bind to different domains of the cloned k opioid receptor. Proc Natl Acad Sci USA 1994 91 8042-8046. 

Mansour A, Fox CA, Burke S, Akil H, Watson SJ. 1995a. Immunohistochemical localization of the cloned mu opioid receptor in the rat CNS. J Chem Neuroanatomy 8 ... 

Progress in the molecular characterization of opioid receptors has been slower than for other cell-surface receptors and, to date, none has been sequenced or cloned and the second messengers mediating opioid actions are still unknown. The literature in this area has been reviewed in 1990 by Lo and Smith [11], who cite three main problems with the opioid receptor it is difficult to solubilize, there are no simple biochemical assays to test the functional integrity of an isolated receptor extract and there are at least three receptor subtypes (designated as mu, kappa and delta). 

It is generally accepted that three major classes of opioid receptors exist. The most commonly used terminology discriminates between p-, K-, and 8-opioid receptors. However, after cloning of the three genes encoding opioid receptors, a new nomenclature was agreed upon OPl (corresponding to the 8 receptor), OP2 (k receptor) and OP3 (p receptor) (Dhawan et ah. 

As noted previously, three major classes of opioid receptors ( -, 5, and ) have been identified in various nervous system sites and in other tissues (Table 31-1). Each of the three major receptors has now been cloned. All are members of the G protein-coupled family of receptors and show significant amino acid sequence homologies. Multiple receptor subtypes have been proposed based on pharmacologic criteria, including 14, v-2, 1, 52 and k2, and k3. However, genes encoding... 

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. 

A second strategy used cloning by homology. Although this approach undoubtedly identified opioid receptor clones, it did not reveal the identity of opioid receptors and claims were retrospective. The approach was based on the assumption that the opioid receptors would be G protein-coupled receptors, and as indicated above, earlier pharmacological evidence strongly... 

The fourth approach, which eventually proved successful, was expression cloning. In this strategy cDNA from a tissue or cell line expressing the receptor was cloned into a mammalian expression vector and transfected in to a cell line devoid of opioid receptors. Transfected cells could then be screened... 

TABLE 3 Relative Efficacies of Biphalin and Delta-Selective Agonists at the Wild-Type Cloned Human Opioid Receptor... 

These discoveries were followed by the isolation of cDNA for two opioid receptors derived from rat brain, identified as ROR-A and ROR-B [55], Competition studies performed with DSLET, DPDPE, and DAMGO in transfected CHO cells determined that the ROR-A corresponds to the 6-opioid receptor and ROR-B corresponds to the p-opioid receptor [55]. Finally, human cDNA for the 6-opioid was expressed in COS-7 cells [56]. Again, competition studies confirmed the presence of the 6-opioid receptor. Moreover, it was determined that the cloned receptor represents the 62-opioid receptor, since NTB showed an approximately eightfold greater affinity than BNTX [56]. The molecular biology of the cloned opioid receptors has since been reviewed [57]. It has been subsequently determined that the cloned 6-opioid receptor may correspond to the 62-isoform [57]. 

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