Opiates such as morphine are the choice analgesic in the treatment of chronic pain. However their long-term use is limited because of the development of tolerance and dependence. Due to its importance in therapy, different strategies have been considered for making opiates such as morphine more effective, while curbing its liability to be abused. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular, ␦ opioid receptor ligands have been useful in enhancing morphine's potency. The underlying molecular basis for these observations is not understood. We propose the modulation of receptor function by physical association between and ␦ opioid receptors as a potential mechanism. In support of this hypothesis, we show that -␦ interacting complexes exist in live cells and native membranes and that the occupancy of ␦ receptors (by antagonists) is sufficient to enhance opioid receptor binding and signaling activity. Furthermore, ␦ receptor antagonists enhance morphine-mediated intrathecal analgesia. Thus, heterodimeric associations between -␦ opioid receptors can be used as a model for the development of novel combination therapies for the treatment of chronic pain and other pathologies. Opioid receptors belong to the rhodopsin family of G proteincoupled receptors (GPCRs). Like many GPCRs, these receptors were thought to function as single units. This notion has been revised in recent years by a number of studies showing that GPCRs associate with each other to form dimers and͞or oligomers (1-3). Of particular significance are the studies with rhodopsin, a prototypical member of the GPCR family, where infrared-laser atomic-force microscopy of native mouse disk membranes showed the receptors to be arranged in crystalline arrays of dimeric units (4, 5). Also, data from x-ray crystallographic studies with rhodopsin (6, 7) and the N terminus of metabotropic glutamate receptors (8), support the notion that dimerization is an integral feature of these receptors and could play a key role in modulating their function.The three types of opioid receptors (, ␦, and ) have been shown to associate with each other in a homotypic or heterotypic fashion when expressed in heterologous cells (9-11). Furthermore, heterotypic interactions appear to alter the ligand-binding and signaling properties of these receptors (12). However, until now, it was not clear whether these interactions occurred in live cells and in endogenous tissues and whether they were physiologically relevant. In this study, we addressed these questions by using multiple approaches. We used the bioluminescence resonance energy transfer (BRET) assay to show that and ␦ receptors interact in living cells. In addition, we show that signaling by clinically relevant drugs, such as morphine, fentanyl, and methadone can be enhanced by ␦ receptor ligands. This potentiation of receptor signaling by the ␦ receptor antagonist is seen in membranes from WT mice and not in membranes from ␦ receptor lacking mice (␦ k͞o). Finally, w...

Adrenergic and opioid receptors belong to the rhodopsin family of G-protein coupled receptors, couple to analogous signal transduction pathways, and affect the nociceptive system. Although a number of previous studies have reported functional interactions between these two receptors, the basis for this has not been well explored. We propose that direct receptor-receptor interactions could account, in part, for opioid-adrenergic cross-talk. In this report, we have addressed this using biophysical, biochemical, and pharmacological studies. We show that opioid and ␣ 2A adrenergic receptors reside in close proximity in live cells using the bioluminescence resonance energy transfer assay. These receptors colocalize to proximal dendrites in primary hippocampal neurons. -␣ 2A Receptor complexes can be isolated from heterologous cells or primary neurons coexpressing these receptors. In these cells, the activation of either or ␣ 2A receptor leads to a significant increase in the level of immunoprecipitable -␣ 2A complexes, whereas activation of both receptors leads to a significant decrease. The implications of these effects on signaling were examined using the agonist-mediated increase in G-protein activity and mitogen-activated protein kinase activity. We find that activation of either or ␣ 2A receptors leads to an increase in the extent of signaling, whereas activation of both receptors leads to a decrease. The increase in signaling by individual ligands and decrease by a combination of ligands is also seen in primary spinal cord neurons endogenously expressing these receptors. Taken together, these results suggest that physical associations between and ␣ 2A receptors could play a role in the functional interactions between these receptors.

Transcription elongation by RNA polymerase II (RNAPII) is negatively regulated by the human factors DRB-sensitivity inducing factor (DSIF) and negative elongation factor (NELF). A 66-kilodalton subunit of NELF (NELF-A) shows limited sequence similarity to hepatitis delta antigen (HDAg), the viral protein required for replication of hepatitis delta virus (HDV). The host RNAPII has been implicated in HDV replication, but the detailed mechanism and the role of HDAg in this process are not understood. We show that HDAg binds RNAPII directly and stimulates transcription by displacing NELF and promoting RNAPII elongation. These results suggest that HDAg may regulate RNAPII elongation during both cellular messenger RNA synthesis and HDV RNA replication.

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RNA polymerase II is implicated in the RNA-templated RNA synthesis during replication of viroids and Hepatitis Delta Virus (HDV); however, neither the RNA template nor protein factor requirements for this process are well defined. We have developed an in vitro transcription system based on HeLa cell nuclear extract (NE), in which a segment of antigenomic RNA corresponding to the left-hand tip region of the HDV rod-like structure serves as a template for efficient and highly specific RNA synthesis. Accumulation of the unique RNA product is highly sensitive to a-amanitin in HeLa NE and only partially sensitive to this drug in NE from PMG cells that contain an allele of the a-amanitinresistant subunit of pol II, strongly suggesting pol II involvement in this reaction. Detailed analysis of the RNA product revealed that it represents a chimeric molecule composed of a newly synthesized transcript covalently attached to the 59 half of the RNA template. Selection of the start site for transcription is remarkably specific and depends on the secondary structure of the RNA template, rather than on its primary sequence. Some features of this reaction resemble the RNA cleavage-extension process observed for pol II-arrested complexes in vitro. A possible involvement of the described reaction in HDV replication is discussed.

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