The δ‐opioid receptor is known to regulate multiple effectors in various tissues. When expressed in human embryonic kidney 293 cells, the cloned δ‐opioid receptor inhibited cyclic AMP (cAMP) accumulation in response to the δ‐selective agonist [d‐Pen2,d‐Pen5]enkephalin. The inhibitory response of [d‐Pen2,d‐Pen5]enkephalin was dependent on the expression of the δ‐opioid receptor and exhibited an EC50 of 1 nM. The receptor showed ligand selectivity and a pharmacological profile that is appropriate for the δ‐opioid subtype. The inhibition was blocked by the opiate antagonist naloxone or by pretreatment of the cells with pertussis toxin. Cotransfection of the δ‐opioid receptor with type II adenylyl cyclase and an activated mutant of αs converted the δ‐opioid signal from inhibition to stimulation of cAMP accumulation. It is interesting that when transfected into Ltk− fibroblasts, the cloned δ‐opioid receptor was able to stimulate the formation of inositol phosphates (EC50 = 8 nM). This response was sensitive to pertussis toxin. The opioid‐mediated formation of inositol phosphates exhibited the same ligand selectivity as seen with the inhibition of cAMP accumulation. The ability of the δ‐opioid receptor to couple to G proteins other than Gi was also examined. Cotransfection studies revealed that the δ‐opioid receptor can utilize Gz to regulate cAMP accumulation and to stimulate the formation of inositol phosphates.
Many Gi-coupled receptors are known to interact with the pertussis toxin (PTX)-insensitive Gz protein. Given that the alpha subunits of Gi and Gz share only 60% identity in their amino acid sequences, their receptor-interacting domains must be highly similar. By swapping the carboxyl termini of alpha i2 and alpha z with each other or with those of alpha t, alpha12, and alpha13, we examined the relative contributions of the carboxyl-end 36 amino acids of the alpha chains toward receptor recognition. Chimeric alpha chains lacking the site for PTX-catalyzed ADP-ribosylation were coexpressed with the type II adenylyl cyclase (AC II) and one of several Gi-coupled receptors (formyl peptide, dopamine D2, and delta-opioid receptors) in human embryonic kidney 293 cells. The alpha i2/alpha z chimera was able to interact with both aminergic and peptidergic receptors, resulting in betagamma-mediated stimulation of AC II in the presence of agonists and PTX. Functional and mutational analyses of alpha i2/alpha z revealed that this chimera can inhibit the endogenous ACs of 293 cells. Similarly, the alpha z/alpha i2 chimera seemed to retain the abilities to interact with receptors and inhibit cAMP accumulation. Fusion of the carboxyl-terminal 36 amino acids of alpha z to a backbone of alpha t1 produced a chimera, alpha t1/alpha z, that did not couple to any of the Gi-coupled receptors tested. Interestingly, an alpha13/alpha z chimera (with only the last five amino acids switched) displayed differential abilities to interact with receptors. Signals from aminergic, but not peptidergic, receptors were transduced by alpha13/alpha z. A similar construct, alpha12/alpha z, behaved just like alpha13/alpha z. These results indicated that "alpha i-like" or "alpha z-like" sequences at the carboxyl termini of alpha subunits are not always necessary or sufficient for specifying interaction with Gi-coupled receptors.
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