Duane E. Keith

Morphine Activates Opioid Receptors without Causing Their Rapid Internalization

We have examined the endocytic trafficking of epitope-tagged δ and μ opioid receptors expressed in human embryonic kidney (HEK) 293 cells. These receptors are activated by peptide agonists (enkephalins) as well as by the alkaloid agonist drugs etorphine and morphine. Enkephalins and etorphine cause opioid receptors to internalize rapidly (t1/2 ~ 6 min) by a mechanism similar to that utilized by a number of other classes of receptor, as indicated by localization of internalized opioid receptors in transferrin-containing endosomes and inhibition of opioid receptor internalization by hypertonic media. Remarkably, morphine does not stimulate the rapid internalization of either δ or μ opioid receptors, even at high concentrations that strongly inhibit adenylyl cyclase. These data indicate that agonist ligands, which have similar effects on receptor-mediated signaling, can have dramatically different effects on the intracellular trafficking of a G protein-coupled receptor.

Sequestration of the δ Opioid Receptor ROLE OF THE C TERMINUS IN AGONIST-MEDIATED INTERNALIZATION

The primary structure of the opioid receptors have revealed that many of the structural features that are conserved in other G protein-coupled receptors are also conserved in the opioid receptors. Upon exposure to agonists, some G protein-coupled receptors internalize rapidly, whereas other structurally homologous G protein-coupled receptors do not. It is not known whether opioid receptors are regulated by rapid endocytosis. In transfected Chinese hamster ovary cells expressing the epitope-tagged wild type δ opioid receptor, exposure to 100 nM [D-Ala2,D-Leu5]enkephalin causes internalization of the receptor within 30 min as determined by confocal microscopy. The rate of internalization of the wild type receptor is rapid with a half-maximal reduction by about 10 min, as determined by the reduction in mean surface receptor fluorescence intensity measured using flow cytometry. In contrast, the cells expressing receptors lacking the C-terminal 15 or 37 amino acids exhibit a substantially slower rate of internalization. Furthermore, the cells expressing receptors with point mutations of any of the Ser/Thr between Ser344 and Ser363 in the C-terminal tail exhibit a significant reduction in the rate of receptor internalization. These results suggest that a portion of the C-terminal tail is involved in receptor internalization. Agents that block the formation of clathrin-coated pits considerably reduce the extent of agonist-mediated internalization of the wild type receptor. Taken together, these results suggest that the wild type opioid receptor undergoes rapid agonist-mediated internalization via a classic endocytic pathway and that a portion of the C-terminal tail plays an important role in this internalization process.

Multiple opioid receptor-like genes are identified in diverse vertebrate phyla

Polymerase chain reaction was used to determine whether opioid receptor‐like sequences are present in species from the protostome and deuterostome branches of the metazoan kingdom. Multiple opioid receptor‐like sequences were found in all vertebrates, but no specific fragments were obtained from any invertebrates. Delta, mu, kappa and ORL‐1 receptors were identified from bovine DNA, and three different opioid receptor‐like fragments were identified from the other vertebrates analyzed. The data suggest that the opioid receptor gene family has been highly conserved during vertebrate evolution and that, even in the primitive jawless fish, multiple members of the opioid receptor family appear to be present.

Mu opioid receptor-like sequences are present throughout vertebrate evolution

The sequence of the mu opioid receptor is highly conserved among human, rat, and mouse. In order to gain insights into the evolution of the mu opioid receptor, polymerase chain reaction (PCR) was used to screen genomic DNA from a number of different species using degenerate oligonucleotides which recognize a highly conserved region. DNA was assayed from representative species of both the protostome and deuterostome branches of the metazoan phylogenetic tree. Mu opioid receptor-like sequences were found in all vertebrate species that were analyzed. These species included bovine, chicken, bullfrog, striped bass, thresher shark, and Pacific hagfish. However, no mu opioid receptor-like sequences were detected from protostomes or from any invertebrates. The PCR results demonstrate that the region of the mu opioid receptor gene between the first intracellular loop and the third transmembrane domain (TM3) has been highly conserved during evolution and that mu opioid receptor-like sequences are present in the earliest stages of vertebrate evolution. Additional opioid receptor-like sequence was obtained from mRNA isolated from Pacific hagfish brain using rapid amplification of cDNA ends (RACE). The sequence of the Pacific hagfish was most homologous with the human mu opioid receptor (72% at the amino acid level between intracellular loop 1 and transmembrane domain 6) although over the same region high homology was also observed with the delta opioid receptor (69%), the kappa receptor (63%), and opioid receptor-like (ORLI) (59%). The hag-fish sequence showed low conservation with the mammalian opioid receptors in the first and second extracellular loops but high conservation in the transmembrane and intracellular domains.