Many cell surface receptors are coupled to the activation or inhibition of intracellular enzymes or ion channels by GTP-binding regulatory proteins (G proteins). Opiate receptors are members of the large class of seven transmembrane spanning helical proteins that are coupled to G proteins. We seek to understand in detail the mechanism of action of receptors for the opioid peptides and their close relatives. The neuroblastoma x glioma hybrid cell line, NG108-15, is richly endowed with opiate receptors, and is a particularly good source of this protein since it expresses only the delta type of opiate receptor. We and others, have cloned the delta opiate receptor of NG108-15 cells, which is the best characterized of this class. In the brain, the gene is expressed at low levels in many regions but transcripts are found in particularly large amounts in the anterior pituitary and pineal glands. Since these tissues are located outside the blood brain barrier, opioid peptides easily can reach receptors in these areas from the blood. The opiate receptors in bovine and rat pineal are almost completely occupied with natural ligands, presumably peptides, but when these are removed a large number of receptors is measured. Opiate antagonists decrease the rate of production of melatonin by pineal cells. Since this process is known to be cAMP dependent, we searched for and obtained evidence for an increase in cAMP levels in pineal cell membranes caused by stimulation of their opiate receptors. This is a highly unusual activity for an opiate receptor which, in most tissues, reduce cAMP levels by inhibiting adenylyl cyclase. We found that this unique result to be mediated by a pertussis toxin sensitive Gi, presumably mediated by a direct G protein beta-gamma-subunit interaction with the an isoform of the enzyme. In the past year we have completed the characterization of eight mutated receptors in which the four conserved proline residues have been replaced by alanine and leucine residues. The proline at position 182, in the fourth transmembrane helix, is particularly interesting in that its substitution by leucine leads to hyperexpression of the mutant receptor in recpient HEK cells and to the appearance of a marked degree of inverse agonist activity, where none previously existed, with suitable peptide ligands. Thus, both drug efficacy and receptor expression are markedly changed by the appropriate modification of the transmembrane helices. - opiate receptors, G proteins, adenylyl cyclase, ion channels, opioid peptides, NG108-15
