Ping Y. Law

Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned μ, δ, and κ opioid receptors

Abstract A rat brain cDNA library was screened for clones homologous to the recently cloned mouse δ-opioid receptor (DOR-1). Among the clones isolated was Hyp 8-1, a clone with a unique nucleotide sequence capable of encoding a putative protein which is 57–58% identical to the amino acid sequences of the cloned δ, μ and κ opioid receptors, indicating a close relationship of Hyp 8-1 with the opioid receptor family. Several cDNAs representing possible splice variants of Hyp 8-1 were also isolated. Binding studies of COS-7 cells transfected with clone Hyp 8-1 failed to demonstrate specific binding with several 3H-opioid ligands. In situ hybridization studies indicate that the mRNA for Hyp 8-1 is distributed discretely throughout the rat brain, in an overall pattern which is different from that of several other G-protein-coupled seven transmembrane receptors. Thus, it is likely that the Hyp 8-1 cDNA encodes a novel peptide receptor.

Inhibition of PI3K/Akt signaling: an emerging paradigm for targeted cancer therapy.

The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB) signaling pathway plays a critical role in cell growth and survival. Dysregulation of this pathway has been found in a variety of cancer cells. Recently, constitutively active PI3K/Akt signaling has been firmly established as a major determinant for cell growth and survival in an array of cancers. Blocking the constitutively active PI3K/AKT signaling pathway provides a new strategy for targeted cancer therapy. Thus, inhibitors of this signaling pathway would be potential anticancer agents, particularly for cancer cells whose survival and growth are dominated by constitutively active PI3K/Akt signaling. This review describes the current understanding of small molecule drugs targeting this pathway both in vitro and in vivo. Inhibitors and functions of the upstream and downstream molecular targets of the PI3K/Akt pathway are discussed in the context of using the inhibitors to block this pathway for targeted cancer therapy. Special emphasis is placed on the following targets: receptor tyrosine kinases, PI3K, Akt, and the mammalian target of rapamycin. While the molecular therapeutic strategy holds great promise for the treatment of a variety of cancers, few small molecule inhibitors with potential high therapeutic indexes are available. Thus, new inhibitors with high selectivity, bioavailability, and potency are greatly needed. Novel approaches toward the development of PI3K/Akt pathway inhibitors as anticancer therapeutics are discussed in detail, with emphasis on chemical genetics-based and structure-based drug design.

Distribution of neuropeptide receptors: New views of peptidergic neurotransmission made possible by antibodies to opioid receptors

The cloning of receptors for neuropeptides made possible studies that identified the neurons that utilize these receptors. In situ hybridization can detect transcripts that encode receptors and thereby identify the cells responsible for their expression, whereas immunocytochemistry enables one to determine the region of the plasma membrane where the receptor is located. We produced antibodies to portions of the predicted amino acid sequences of delta, mu, and kappa opioid receptors and used them in combination with antibodies to a variety of neurotransmitters in multicolor immunofluorescence studies visualized by confocal microscopy. Several findings are notable: First, the cloned delta opioid receptor appears to be distributed primarily in axons, and therefore most likely functions in a presynaptic manner. Second, the cloned mu and kappa opioid receptors are found associated with neuronal plasma membranes of dendrites and cell bodies and therefore most likely function in a postsynaptic manner. However, in certain, discrete populations of neurons, mu and kappa opioid receptors appear to be distributed in axons. Third, enkephalin-containing terminals are often found in close proximity (although not necessarily synaptically linked) to membranes containing either the delta or mu opioid receptors, whereas dynorphin-containing terminals are often found in proximity to kappa opioid receptors. Finally, a substantial mismatch between opioid receptors and their endogenous ligands was observed in some brain regions. However, this mismatch was characterized by complementary zones of receptor and ligand, suggesting underlying principles of organization that underlie long-distance, nonsynaptic neurotransmission.

Visualization of time-dependent redistribution of δ-opioid receptors in neuronal cells during prolonged agonist exposure

To date, the visualization of delta-opioid receptor (DOR) internalization has been largely focused on the events of short-term agonist treatment in transfected non-neuronal cells. In this study, we followed DOR trafficking upon prolonged agonist exposure in the neuronally derived neuro2a cells, stably transfected with the fusion DOR (HA-DOR) cDNA. Internalization of surface DOR was clearly visualized in 5 min of exposure to agonist (100 nM DADLE), and the cell surface DOR remained low throughout the entire 24 h agonist exposure. Significant intracellular accumulation was visible at 20 min exposure, and increased to a maximum at 4 h, after which intracellular DOR staining gradually diminished. DOR intracellular staining was enhanced in the presence of agonist and chloroquine, a lysosomotropic agent, suggesting that internalized receptors were targeted to lysosomes and degraded upon prolonged treatment. Time-dependent colocalization of DOR with transferrin and LAMP-2 following short-term and prolonged agonist exposure further confirmed that receptor was distributed to early endosomes (sequestration) and subjected to lysosomes for degradation (down-regulation), respectively.

Properties of a κ-opioid receptor expressed in CHO cells : interaction with multiple G-proteins is not specific for any individual Gα subunit and is similar to that of other opioid receptors

The purpose of the present study was to examine the coupling pattern of a recently cloned kappa-opioid receptor stably transfected in CHO cells to individual G alpha subunits with subsequent comparison to that observed previously for delta- and mu-opioid receptors. Data presented in the current study indicate the successful stable expression of a kappa-opioid receptor in CHO cells. This is supported by experiments in which ligands with selectivity for kappa-, but not delta- or mu-opioid receptors demonstrated high affinity for the expressed receptor and were able to potently and efficaciously produce inhibition of adenylyl cyclase activity. In addition, only kappa-opioid agonists were able to induce dose-dependent increases in the incorporation of [32P]azidoanilido-GTP into four G alpha subunits, three of which were identified as Gi3 alpha, Gi2 alpha and Go2 alpha. Further, the amount of kappa-opioid agonists required to induce 50% maximal labeling of any individual G alpha subunit was similar. Although kappa-opioid agonists produced equivalent maximal labelling of Gi3 alpha, Gi2 alpha and Go2 alpha, significantly less agonist-induced labeling was observed for an unknown G-protein designated as G? alpha. Although these results are slightly different than those observed previously for both delta- and mu-opioid receptors, it appears that all opioid receptors stably transfected in CHO cells interact with multiple G-proteins and that this coupling is not selective for any individual G alpha subunit.

Role of carboxyl terminus of μ- and δ-opioid receptor in agonist -induced down-regulation

Chronic exposure of μ- and δ-opioid receptors to their agonists leads to different rates in receptor down-regulation. In order to analyze the role of the carboxyl terminus of μ- and δ-opioid receptors in the difference in the rate of down-regulation, two chimeras of these receptors were generated by swapping the carboxyl termini; MORTAGDT and DORTAGMT. These chimeras were tagged at the N-terminus with hemagglutinin (HA) epitope (YPYDVPDYA), which can be recognized by the monoclonal antibody 12CA5, and then stably expressed in Neuro 2A (N2A) cells. The swapping of the carboxyl termini did not alter the ligand selectivity of these receptor chimeras. However, they did exhibit a reduction in agonist potency to inhibit forskolin-stimulated adenylyl cyclase activity for all agonists tested except etorphine which had a potency comparable to that of wild type receptors. Treatment of the N2A cells expressing MORTAGDT with 50 nM etorphine produced a faster rate of receptor down-regulation when compared to the wild type μ-opioid receptor. Immunofluorescence microscopy of the MORTAGDT chimera using a monoclonal antibody against HA confirmed internalization of the receptors after treatment with etorphine for 1 and 6 h. There was a reduction in the HA-immunoreactivity at the cell surface of the MORTAGDT chimera concurrent with more noticeable HA-immunoreactivity inside the cell compared to the wild type receptor. On the other hand, the rate of down-regulation of DORTAGMT receptors was seen to be the same as the wild type δ-opioid receptor after etorphine treatment. Immunofluorescence studies showed more reduction in cell surface staining of the DORTAGMT chimera compared to the wild type receptor. These data suggest the involvement of the carboxyl terminus in agonist-induced down-regulation and internalization of the μ-opioid receptor. However, different mechanisms that are unrelated to the carboxyl terminus may operate in the down-regulation of δ-opioid receptor.

The first and third intracellular loops together with the carboxy terminal tail of the δ-opioid receptor contribute toward functional interaction with Gα16

Opioid peptides exert their regulatory effects on both central and pheripheral nervous systems via multiple opioid receptors that are linked to seemingly identical sets of guanine nucleotide‐binding regulatory proteins (G proteins). In contrast to the μ‐opioid receptor, the δ‐opioid receptor can efficiently stimulate phospholipase C via G16. We used a series of μ/δ‐opioid receptor chimeras to examine the involvement of intracellular receptor domains in the recognition of G16. After ascertaining that the chimeras can bind opioid ligands with high affinity and elicit inhibition of adenylyl cyclase, COS‐7 cells were cotransfected with cDNAs encoding Gα16 and a μ/δ‐opioid receptor chimera and assayed for [d‐Ala2,d‐Leu5]enkephalin‐induced stimulation of phospholipase C. Our results indicate that (i) the carboxy terminal tail of the δ‐opioid receptor is necessary but insufficient for conferring coupling to Gα16, (ii) the third inner loop together with the carboxy terminal tail of the δ‐opioid receptor can provide sufficient contact domains for Gα16, and (iii) the first inner loop of the δ‐opioid receptor, in particular Leu80, as well as the fifth transmembrane domain and/or the third extracellular loop may also contribute in defining the fidelity of interaction between the δ‐opioid receptor and Gα16. These results indicate that efficient coupling of the δ‐opioid receptor to Gα16 requires the participation of most of the intracellular regions, including the first intracellular loop.

Functional expression of adrenergic and opioid receptors in Xenopus oocytes: interaction between α2- and β2-adrenergic receptors

We functionally expressed alpha 2-adrenergic, beta 2-adrenergic, and delta-opioid receptors in Xenopus laevis oocytes. We detected receptor function as changes in currents carried by adenosine 3,5-cyclic monophosphate (cAMP)-regulated chloride channels provided by the cystic fibrosis transmembrane conductance regulator (CFTR) and recorded by two-electrode voltage clamp. Co-application of forskolin and isobutylmethylxanthine (IBMX) or IBMX alone produced currents with a reversal potential indicative of chloride ions only in oocytes previously injected with mRNA encoding CFTR. Isoproterenol produced concentration-dependent responses in oocytes injected with mRNA encoding beta 2-adrenergic receptors and CFTR, and co-administration of propranolol antagonized these responses. Similarly, the alpha 2-adrenergic agonist UK14304 increased IBMX-induced currents only in oocytes injected with mRNA encoding alpha 2-adrenergic receptors and CFTR, and idazoxan antagonized these enhancements. The delta-opioid agonist DADLE produced concentration-related, naloxone-reversible increases in IBMX- and forskolin-induced currents only in oocytes injected with mRNA encoding delta-opioid receptors and CFTR. In oocytes co-injected with alpha 2, beta 2, and CFTR mRNAs, isobolographic analysis revealed an additive interaction between alpha 2- and beta 2-adrenergic receptors. These studies establish the oocyte as a cell system for studying the interactions among cAMP-modulating G protein-coupled receptors and provide another example of alternative coupling of alpha 2-adrenergic and delta-opioid receptors to G proteins, possibly Gs proteins, other than Gi proteins.

Affinity labels as tools for the identification of opioid receptor recognition sites

Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named reporter affinity labels because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.

Dynamic association of p300 with the promoter of the G protein-coupled rat delta opioid receptor gene during NGF-induced neuronal differentiation.

The G protein-coupled delta opioid receptor (DOR) plays a critical role in pain control. Emerging evidence shows that DOR also plays a role in neuronal differentiation and survival. Nerve growth factor (NGF) is known to be critical for the development and maintenance of the central and peripheral nervous systems. Our previous studies have shown that sustained activation of NGF/PI3K/Akt/NF-kappaB signaling is essential for NGF-induced dor gene expression during neuronal differentiation and that the epigenetic modifications at histone 3 lysine 9 temporally correlate with the dor gene transcription. In this study, we cloned the rat dor gene promoter and identified an NGF-responsive region similar to that from the mouse dor gene promoter. We further identified p300, a known NF-kappaB binding partner with intrinsic histone acetyltransferase activity, to be dynamically associated with the dor gene. We also found that assembling of RNA polymerase II (Pol II) at the promoter took place before NGF stimulation, indicating that p300 could only interact with preassembled Pol II at the promoter after NGF stimulation. Taken together, these results implicate that preassembly of the Pol II preinitiation complex, sustained activation of PI3K/Akt/NF-kappaB signaling, and dynamic p300 association at the promoters sequentially is one of the mechanisms of induction of the late phase genes during NGF-induced neuronal differentiation.