Opiate cloning

Opiates iateract with three principal classes of opioid GPCRs )J.-selective for the endorphiQS,5-selective for enkephalins, and K-selective for dynorphias (51). AU. three receptors have been cloned. Each inhibits adenylate cyclase, can activate potassium channels, and inhibit A/-type calcium channels. The classical opiates, morphine and its antagonists naloxone (144) and naltrexone (145), have moderate selectivity for the. -receptor. Pharmacological evidence suggests that there are two subtypes of the. -receptor and three subtypes each of the 5- and K-receptor. An s-opiate receptor may also exist. 

SKIP produces its effects through two classes of GPCR, SRIF-1 and SRIF-2 that are structurally related to cloned opiate receptors. The agonists,... 

The k receptor was cloned by a completely different approach. Yasuda et al. [9] employed probes against conserved regions of somatostatin receptors to screen a mouse brain cDNA library. Mouse k and 3 receptor cDNAs were isolated using this procedure that established that opiate and somatostatin receptors have high amino acid sequence similarity, consistent with their ability to bind some common ligands, such as Sandostatin. 

The pharmacological properties of the cloned opiate receptors are similar to the characteristics of the endogenously expressed receptors [9, 36, 45]. The binding of opiates to the cloned receptors is stereoselective and the antagonist naloxone interacts with all three of the cloned receptors, although naloxone had much lower af-... 

The cloned opiate receptors, like the endogenously expressed receptors, can couple to phospholipase C to increase Ca++ mobilization in transfected cells [31, 57, 58]. Thus, stimulation of opiate receptors can lead to inhibition of Ca++ influx via voltage-sensitive channels as well as increase Ca++ levels in cells released from intracellular stores. This dual role of opiates may explain the opposing excitatory and inhibitory effects they may have on some cells in the nervous system [59]. 

The cloned opiate receptors expressed in homogeneous cell lines associate with guanine nucleotide binding (G) proteins which couple the receptors to cellular effector systems. G proteins consist of hetero-trimeric complexes of a, fj and y subunits [66]. Multiple subtypes of each subunit have been identified and cloned. In particular, three subtypes of Gi have been cloned that are approximately 90% identical in amino acid sequence. Furthermore, two splice forms of G0 have been identified. 

The multiplicity of G proteins coupled to opiate receptors may explain how different opiates can bind to the same receptor yet induce different cellular responses. For example, morphine binds to the cloned rat fi receptor expressed in HEK 293, CHO and COS-7 cells and inhibits cAMP accumulation [80-82]. Morphine can be continuously applied to the cells for up to 16 h, and the potency and magnitude of morphine inhibition of adenylyl cyclase does not diminish [80, 81]. In contrast, the opiate sufentanil can bind to the same cloned fi receptor in HEK 293 cells to inhibit cAMP accumulation. However, sufentanil s actions rapidly desensitize [83]. Since both compounds bind to the same receptor, and the fi receptor is the only receptor these drugs can interact with in these cells, the ability of these two full agonists to differentially regulate the fi receptor must be due to their abilities to affect separate adaptive processes in these cells. 

The desensitization of the fi receptor was heterologous. In oocytes cotransfected with ft and serotonin receptors, chronic morphine treatment abolished morphine and serotonin potentiation of the K+ current [63]. Similarly, in AtT-20 cells transfected with the cloned fi receptor, chronic DAMGO treatment abolished the ability of opiates and somatostatin, acting via endogenous somatostatin receptors in these cells, to stimulate K+ conductance [65]. 

Structure-Function Analysis of the Cloned Opiate Receptors... 

Changing the amino acid sequence of the cloned receptors by mutating nucleotides within the receptor cDNAs has proven to be an effective mechanism by which to identify structural features of the receptors responsible for their unique functional properties. In particular, site-directed mutagenesis has been employed to determine the ligand binding domains of each opiate receptor. 

Blake AD, Bot G, Reisine T. Structure-function analysis of the cloned opiate receptors peptide and small molecule interactions. Chem Biol 1996 3 967-972. 

Wang J, Johnson P, Persico Am Hawkins A, Griffin C, Uhl G. Human mu opiate receptor cDNA and genomic clones, pharmacological characterization and chromosomal assignment. FEBS Lett 1994 338 217-222. 

Another important but infrequent modification of peptides is a-N-acetylation (Figs 18-5,18-7). During POMC processing, a-N-acetylation greatly increases the skin-darkening potency of ACTH(1-13)NH2 while abolishing both the adrenal steroidogenic potency of ACTH and the opiate activity of (3-endorphin [2]. The enzyme(s) responsible for this modification has not yet been purified or cloned. 

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