Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression.

Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.

Differential binding properties of [3H]dextrorphan and [3H]MK-801 in heterologously expressed NMDA receptors.

The N-methyl-D-aspartate receptor (NMDAR) antagonists: MK-801, phencyclidine and ketamine are open-channel blockers with limited clinical value due to psychotomimetic effects. Similarly, the psychotomimetic effects of the dextrorotatory opioids, dextromethorphan and its metabolite dextrorphan, derive from their NMDAR antagonist actions. Differences in the use dependency of blockade, however, suggest that the binding sites for MK-801 and dextrorphan are distinct. In the absence of exogenous glutamate and glycine, the rate of association of [3H]MK-801 with wild-type NR1-1a/NR2A receptors was considerably slower than that for [3H]dextrorphan. Glutamate individually, and in the presence of the co-agonist glycine, had substantial effects on the specific binding of [3H]MK-801, while the binding of [3H]dextrorphan was not affected. Mutation of residues N616 and A627 in the NR1 subunit had a profound effect on [3H]MK-801 binding affinity, while that of [3H]dextrorphan was unaltered. In contrast, NR1 residues, W611 and N812, were critical for specific binding of [3H]dextrorphan to NR1-1a/NR2A complexes with no corresponding influence on that of [3H]MK-801. Thus, [3H]dextrorphan and [3H]MK-801 have distinct molecular determinants for high-affinity binding. The ability of [3H]dextrorphan to bind to a closed channel, moreover, indicates that its recognition site is shallower in the ion channel domain than that of MK-801 and may be associated with the extracellular vestibule of the NMDAR.

Synthesis and evaluation of N,N-dialkyl enkephalin-based affinity labels for delta opioid receptors.

To develop affinity labels for delta opioid receptors based on peptide antagonists, the Phe(4) residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib(2),Aib(3)]leucine enkephalin (ICI-174, 864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH(2)Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH(2)) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing delta or mu opioid receptors. The delta receptor affinities of the N, N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe(4) decreased binding affinity at both delta and mu receptors in standard radioligand binding assays; the one exception was N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for delta receptors (IC(50) = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC(50) = 78.2 nM). The decreases in mu receptor affinities were greater than in delta receptor affinities so that all of the analogues tested exhibited significantly greater delta receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to delta receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of delta opioid receptors.

Synthesis and opioid activity of conformationally constrained dynorphin A analogues. 2. Conformational constraint in the "address" sequence.

Several cyclic lactam analogues of Dyn A-(1-13)NH2 were prepared in order to reduce the conformational flexibility in different regions of the native linear peptide. Cyclo[D-Asp(i),Dap(i+3)]Dyn A-(1-13)NH2 (Dap = alpha,beta-diaminopropionic acid) analogues were designed on the basis of molecular modeling using AMBER, which suggested that this constraint may be compatible with an alpha-helix. The cyclic portion of these constrained analogues spanned from residues 3 to 9, a region proposed by Schwyzer (Biochemistry 1986, 25, 4281) to adopt a helical conformation at kappa receptor sites. Analogues containing Dab (alpha,gamma-diaminobutyric acid) or Orn in position i + 3 were also synthesized to examine the effects of larger ring size. The cyclic peptides exhibited marked differences in binding affinities for kappa, mu, and delta receptors and in opioid activity in the guinea pig ileum (GPI). Cyclo[D-Asp6,Dap9]Dyn A-(1-13)NH2 showed both high kappa receptor affinity and potent agonist activity in the GPI, while cyclo[D-Asp3,Dap6]Dyn A-(1-13)NH2 exhibited very weak binding affinity at all opioid receptors as well as very weak opioid activity in the GPI. Cyclo[D-Asp5,Dap8]Dyn A-(1-13)NH2 showed moderate binding affinity for kappa receptors and was the most kappa selective ligand in this study, but this peptide exhibited very weak agonist activity in the GPI assay. Compared to the corresponding linear peptides, all of the cyclic peptides exhibited decreased mu receptor affinity, while kappa receptor affinity was retained or improved. Therefore the corresponding linear peptides were generally mu selective while the cyclic constrained peptides demonstrated slight selectivity for kappa vs mu receptors or were nonselective. Increasing the ring size by incorporating Dab or Orn in positions 6, 8, or 9 did not significantly affect the binding affinity for the three opioid receptor types nor the opioid activity observed in the GPI. Circular dichroism spectra of the cyclo[D-Asp(i),Dap(i+3)] derivatives in 80% trifluoroethanol at 25 and 5 degrees C suggested differences in the stability of a helical structure when the constraint was incorporated near the N-terminus vs in the middle of the peptide.

Dextrorotatory opioids induce stereotyped behavior in Sprague-Dawley and Dark Agouti rats.

Dextromethorphan and dextrorphan elicited a stereotyped behavioral syndrome in rats indistinguishable from that produced by PCP and other non-competitive NMDA antagonists. The rank order of potency for the induction of stereotyped behavior in male Sprague-Dawley rats was: MK-801>PCP>(+/-)cyclazocine>dextrorphan>(+/-)ketamine>dextromethorphan. These behavioral potencies were significantly correlated (0.91; P<0.05) with their respective affinities for high affinity [3H]dextrorphan-labelled NMDA receptors in rat forebrain membranes. To address the propensity of dextromethorphan to induce stereotyped behavior, dextrorotatory-opioid induced stereotypies were investigated in female Dark Agouti and female Sprague-Dawley rats. The female Dark Agouti lacks CYP2D1, the cytochrome P450 enzyme which catalyses the oxidative O-demethylation of dextromethorphan to dextrorphan. No differences were observed in either potency or time to peak effect for dextromethorphan to induce stereotyped behavior in the rat strains, suggesting that the affinity of dextromethorphan for NMDA receptors adequately accounts for its ability to induce stereotyped behavior. Female Dark Agouti rats were, however, more sensitive to the effects of dextrorphan, which may reflect differences in the ability of this strain to metabolize dextrorphan. We find no evidence to suggest that dextromethorphan produces a behavioral syndrome in rats that is distinct from that induced by dextrorphan. The commonality between the pharmacologic profiles of these compounds suggests that the abuse potential of dextromethorphan containing antitussive preparations is related to the non-competitive NMDA antagonist activity of dextromethorphan and its metabolites.

High level expression of the NMDAR1 glutamate receptor subunit in electroporated COS cells.

The rat N-methyl-D-aspartate (NMDA) glutamate receptor subunit NR1-1a was transiently expressed in COS cells using the technique of electroporation, which was fivefold more efficient than the calcium phosphate precipitation method of transfection. The glycine site antagonist 5,7-[3H]dichlorokynurenic acid labeled a single high-affinity site (KD = 29.6 +/- 6 nM; Bmax = 19.4 +/- 1.6 pmol/mg of protein) in membranes derived from COS cells electroporated with NR1-1a. In contrast to previous reports using transiently transfected human embryonic kidney 293 cells, binding of the noncompetitive antagonist (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]MK-801) was not detected in NR1-1a-transfected COS cells. Although immunofluorescent labeling of electroporated COS cells demonstrated that the NR1-1a protein appears to be associated with the cell membrane, neither NMDA nor glutamate effected an increase in intracellular calcium concentration in fura-2-loaded cells, suggesting that homomeric NR1-1a receptors do not act as functional ligand-gated ion channels. Therefore, COS cells appear to differ from Xenopus oocytes with respect to the transient expression of functional homomeric NR1 receptors. Although expression of NR1-1a is sufficient to reconstitute a glycine binding site with wild-type affinity for antagonists in COS cells, recombinant homomeric NR1-1a receptors do not display properties that are characteristic of native NMDA receptors, such as permeability to Ca2+ and channel occupancy by MK-801, when expressed in this mammalian cell line.

Isolation of a novel family of C(2)H(2) zinc finger proteins implicated in transcriptional repression mediated by chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan nuclear receptors.

Two novel and related C(2)H(2) zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUP TF-interacting proteins 1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of ARP1 was required for interaction with CTIP1. CTIP1, which exhibited a punctate staining pattern within the nucleus of transfected cells, recruited cotransfected ARP1 to these foci and potentiated ARP1-mediated transcriptional repression of a reporter construct. However, transcriptional repression mediated by ARP1 acting through CTIP1 did not appear to involve recruitment of a trichostatin A-sensitive histone deacetylase(s) to the template, suggesting that this repression pathway may be distinct from that utilized by several other nuclear receptors.

p300 functions as a coactivator for the peroxisome proliferator-activated receptor alpha.

The integrator protein, p300, was demonstrated to interact with mouse peroxisome proliferator-activated receptor alpha in a ligand-enhanced manner. The PPARalpha-interacting domain of p300 was mapped to amino acids 39-117 which interacted strongly with PPARalpha but did not interact with retinoic acid receptor-gamma or retinoid X receptor-alpha. Amino acids within the carboxyl terminus of PPARalpha as well as residues within the hinge region were required for ligand-dependent interaction with p300. p300 enhanced the transcriptional activation properties of PPARalpha and, therefore, can be considered a bona fide coactivator for this nuclear receptor. These observations extend the group of p300-interacting proteins to include mPPARalpha and further characterize the molecular mechanisms of PPARalpha-mediated transcriptional regulation.

Identification of nuclear receptor corepressor as a peroxisome proliferator-activated receptor alpha interacting protein.

Nuclear receptor corepressor (NCoR) was demonstrated to interact strongly with peroxisome proliferator-activated receptor alpha (PPARalpha), and PPARalpha ligands suppressed this interaction. In contrast to the interaction of PPARalpha with the coactivator protein, p300, association of the receptor with NCoR did not require any part of the PPARalpha ligand binding domain. NCoR was found to suppress PPARalpha-dependent transcriptional activation in the context of a PPARalpha.retinoid X receptor alpha (RXRalpha) heterodimeric complex bound to a peroxisome proliferator-responsive element in human embryonic kidney 293 cells. This repression was reversed agonists of either receptor demonstrating a functional interaction between NCoR and PPARalpha.RXRalpha heterodimeric complexes in mammalian cells. NCoR appears to influence PPARalpha signaling pathways and, therefore, may modulate tissue responsiveness to peroxisome proliferators.

Heterodimeric interactions between chicken ovalbumin upstream promoter-transcription factor family members ARP1 and ear2.

Members of the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) subfamily of orphan nuclear receptors, which minimally includes COUP-TFI and ARP1, are highly expressed in brain and are generally considered to be constitutive repressors of transcription. We have used a yeast two-hybrid system to isolate proteins expressed in brain that interact with ARP1. One of the proteins isolated in this screen was Ear2, another orphan receptor that has been suggested to be a member of the COUP-TF subfamily. Here we demonstrate that ARP1 and Ear2 form heterodimers in solution and on directly repeated response elements with high efficiency and a specificity differing from that of homodimeric complexes composed of either receptor. ARP1 and Ear2 were observed to interact in mammalian cells, and the tissue distribution of Ear2 transcripts was found to overlap precisely with the expression pattern of ARP1 in several mouse tissues and embryonal carcinoma cell lines. Heterodimeric interactions between ARP1 and Ear2 may define a distinct pathway of orphan receptor signaling.

Interaction of GRASP, a protein encoded by a novel retinoic acid-induced gene, with members of the cytohesin family of guanine nucleotide exchange factors.

A novel, retinoic acid-induced gene, GRP1-associated scaffold protein (GRASP), was isolated from P19 embryonal carcinoma cells using a subtractive screening strategy. GRASP was found to be highly expressed in brain and exhibited lower levels of expression in lung, heart, embryo, kidney, and ovary. The predicted amino acid sequence of GRASP is characterized by several putative protein-protein interaction motifs, suggesting that GRASP may be a component of a larger protein complex in the cell. Although GRASP does not harbor a predicted membrane spanning domain(s), the protein was observed to be associated with the plasma membrane of transiently transfected mammalian cells. Yeast two-hybrid screening revealed that GRASP interacted strongly with the General Receptor for Phosphoinositides 1 (GRP1), a brefeldin A-insensitive guanine nucleotide exchange factor for the ADP-ribosylation factor family of proteins. GRASP. GRP1 interactions were also demonstrated in vitro and in mammalian cells in which GRASP was shown to enhance GRP1 association with the plasma membrane. Furthermore, GRASP colocalized with endogenous ADP-ribosylation factors at the plasma membrane in transfected cells, suggesting that GRASP may modulate signaling by this family of small GTPases.