Infrequent alterations of the RAR alpha gene in acute myelogenous leukemias, retinoic acid-resistant acute promyelocytic leukemias, myelodysplastic syndromes, and cell lines

Retinoids are important regulators of cell growth and differentiation in vitro and in vivo and they exert their biologic activities by binding to nuclear retinoic acid receptors (RARs; alpha, beta, and gamma) and retinoid X receptors (RXRs; alpha, beta, and gamma). All-trans retinoic acid (RA) induces complete remission in patients with acute promyelocytic leukemia (APL) presumably by binding directly to RAR alpha of APL cells. Leukemic blasts from APL patients initially responsive to RA can become resistant to the agent. HL-60 myeloblasts cultured with RA have developed mutations of the ligand-binding region of RAR alpha and have become resistant to RA. Furthermore, insertion of an RAR alpha with an alteration in the ligand-binding region into normal murine bone marrow cells can result in growth factor-dependent immortalization of the early hematopoietic cells. To determine if alterations of the ligand binding domain of RAR alpha might be involved in several malignant hematologic disorders, the mutational status of this region (exons 7, 8, and 9) was examined in 118 samples that included a variety of cell lines and fresh cells from patients with myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), including 20 APL patients, 5 of whom were resistant to RA and 1 who was refractory to RA at diagnosis, using polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis and DNA sequencing. In addition, 7 of the 20 APLs were studied for alterations of the other coding exons of the gene (exons 2 through 6). No mutations of RAR alpha were detected. Although the sensitivity of PCR-SSCP analysis is less than 100%, these findings suggest that alterations of RAR alpha gene are rare and therefore other mechanisms must be involved in the onset of resistance to retinoids and in the lack of differentiation in disorders of the myeloid lineage.     

Genetic control of the development by retinoic acid

Two families of nuclear receptors for retinoic acid (RA) have been characterized. Members of the RAR family (types alpha, beta and gamma and their isoforms alpha 1, alpha 2, beta 1 to beta 4, and gamma 1 and gamma 2) are activated by most physiologically occurring retinoids (all-trans RA, 9-cis RA, 4oxo RA and 3,4 dihyroRA). In contrast, members of the RXR family (types alpha, beta and gamma and their isoforms) are activated by 9cis-RA only. In addition to the multiplicity of receptors, the complexity of retinoid signalling is further increased by the fact that, at least in vitro, RARs bind to their cognate response elements as heterodimers with RXRs. Moreover, RXRs can also bind, in vitro, to some DNA elements as homodimers, and are heterodimeric partners for other nuclear receptors, including TRs, VDR, PPARs and a number of orphan nuclear receptors. To evaluate the functions of the different RARs and RXRs types and isoforms, we have generated null mutant mice by targeted gene disruption in ES cells. As to the functions of RARs, we found that RAR alpha 1 and RAR gamma 2 null mutant mice are apparently normal. Mice deficient in RAR alpha or RAR gamma (i.e., all alpha or gamma isoforms disrupted) show aspects of the post-natal vitamin A deficiency (VAD) syndrome which can be cured or prevented by RA, including post-natal lethality, poor weight gain and male sterility. RAR beta 2 (and RAR beta) null mutants display a retrolenticular membrane which represents the most frequent defect of the fetal VAD syndrome. That these abnormalities were restricted to a small subset of the tissues normally expressing these receptors suggested that some degree of functional redundancy should exist in the RAR family. To test this hypothesis we then generated RAR double null mutants. RAR alpha beta, RAR alpha gamma and RAR beta gamma compound mutants exhibit all the malformations of the fetal VAD syndrome, thus demonstrating that RA is the vitamin A derivative which plays a crucial role at many different stages and in different structures during organogenesis. Interestingly, almost all the structures derived from mesenchymal neural crests cells (NCC) are affected in RAR compound mutants. As to the functions of RXRs, RXR gamma null mutants are viable, fertile and morphologically normal. In contrast, RXR alpha null fetuses display a thin ventricular wall and die in utero from cardiac failure. A myocardial hypoplasia has also been observed in some RAR compound mutants as well as in VAD fetuses. Thus, RXR alpha seems to act as an inhibitor of ventricular cardiocyte differentiation and/or as a positive regulator of their proliferation, and these functions might involve heterodimerization with RARs and activation by RA. RXR beta null mutants are viable but the males are sterile, most probably because of an abnormal lipid metabolism in the Sertoli cells. New abnormalities, absent in RXR alpha mutants, are generated in RXR alpha/RAR (alpha, beta or gamma) compound mutants. All these abnormalities are also seen in RAR double mutants as well as in VAD fetuses. In contrast, such manifestations of synergism are not observed between the RXR beta or RXR gamma and the RAR (alpha, beta or gamma) null mutations. These data strongly support the conclusion that RXR alpha/RAR heterodimers represent the main functional units of the RA signalling pathway during embryonic development. Moreover, since RXR gamma-/-/RXR beta-/-/RXR alpha +/-mutants are viable, a single allele of RXR alpha can perform most of the developmental RXR functions.     

Arg278, but not Lys229 or Lys236, plays an important role in the binding of retinoic acid by retinoic acid receptor gamma

The diverse biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, beta and gamma) and retinoid X receptors (RXR alpha, beta, and gamma). Although the ligand-binding domains of RARs share the same novel folding pattern, many RAR subtype-specific retinoids have been synthesized indicating that the ligand-binding pocket of each RAR subtype has unique features. Previously we have demonstrated the importance for RA binding and RA-dependent transactivation of Arg276 of RARalpha alone and in RARbeta Arg269 in conjunction with Lys220. In this study, we have examined the role of the homologous amino acid residues (Lys229 and Arg278) in RARgamma for these activities. Like RARalpha but dissimilar to RARbeta, Arg278 in RARgamma alone was found to play an important role in RA binding and RA-dependent transactivation. Since Lys236 in RARgamma was suggested from the crystal structure of holo-RARgamma to interact with RA, we also examined its role and that of its homologs in RARalpha and RARbeta. Despite the suggestion from the crystal structure, neither Lys236 nor its homologs in RARalpha and RARbeta play a role in the binding of RA or RA-dependent transactivation. It is likely that Lys236 in RARgamma and its homologs in RARalpha and RARbeta are solvent exposed rather than pointing into the RA-binding pocket.     

Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics

The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.     

Dimerization of the head-rod junction of scallop myosin

Retinoic acid (RA) is an important mediator of cell differentiation. It stimulates hCG secretion by JEG-3 choriocarcinoma cells in vitro after a time lag. The first aim of this study was to characterize which types of retinoid receptors (RARs and RXRs) are present in JEG-3 cells. Using Western blot analysis and immunocytochemistry with specific antibodies as well as Northern blot analysis, we found that JEG-3 cells expressed RAR alpha and RXR alpha, the latter being the predominant receptor. We then analyzed the action on cell proliferation and hCG secretion of the physiological retinoids all-trans RA (RA) and 9 cis RA as well as synthetic retinoids with specific affinity for RAR alpha and RXR alpha. All these retinoids were potent inhibitors of cell growth, maximal inhibition (72 +/- 2%) being observed after 4 days of treatment with Ro 25, a RXR alpha specific ligand. Within 24 h, 9 cis RA and Ro 25 stimulated hCG secretion, and maximal stimulation (1,472 +/- 10%) occurred at 48 h with the RXR alpha-specific ligand. The RAR alpha-specific ligand also stimulated hCG secretion but to a lower extend and after a delay of 48 h. These results suggest a predominant role of RXR alpha in mediating the biological effects of retinoids on JEG-3 cells and the possible induction by RA itself of the metabolic pathway leading to 9 cis RA.     

Binding of G protein beta gamma-subunits to pleckstrin homology domains

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.     

Response of four human ovarian carcinoma cell lines to all-trans retinoic acid: relationship with induction of differentiation and retinoic acid receptor expression

The response of 4 human ovarian carcinoma cell lines to retinoic acid was found to be related to the histological type and degree of differentiation of these tumor cells. The 2 serous cell lines NIHOVCAR3 and OVCCR1 were the most sensitive to the antiproliferative effect of RA. This inhibition was associated with morphological and biological changes that were indicative of differentiation. The undifferentiated IGROV1 cell line was not affected by RA. Since the effects of RA are thought to be mediated by nuclear retinoic acid receptors (RARs), the expression of RARs in human ovarian cancer cells was studied. RAR alpha was detected as mRNA species of 3.1 and 2.6 kb in all 4 cell lines. RAR beta was not detected in any of the cell lines, while RAR gamma (3 kb) was expressed in all of the ovarian cancer cells but at a very low level in the RA-resistant IGROV1 cells.     

Differential activity of the G protein beta5 gamma2 subunit at receptors and effectors

The G protein beta5 subunit differs substantially in amino acid sequence from the other known beta subunits suggesting that beta gamma dimers containing this protein may play specialized roles in cell signaling. To examine the functional properties of the beta5 subunit, recombinant beta5 gamma2 dimers were purified from baculovirus-infected Sf9 insect cells using a strategy based on two affinity tags (hexahistidine and FLAG) engineered into the N terminus of the gamma2 subunit (gamma2HF). The function of the pure beta5 gamma2HF dimers was examined in three assays: activation of pure phospholipase C-beta in lipid vesicles; activation of recombinant, type II adenylyl cyclase expressed in Sf9 cell membranes; and coupling of alpha subunits to the endothelin B (ETB) and M1 muscarinic receptors. In each case, the efficacy of the beta5 gamma2HF dimer was compared with that of the beta1 gamma2HF dimer, which has demonstrated activity in these assays. The beta5 gamma2HF dimer activated phospholipase C-beta with a potency and efficacy similar to that of beta1 gamma2 or beta1 gamma2HF; however, it was markedly less effective than the beta1 gamma2HF or beta1 gamma2 dimer in its ability to activate type II adenylyl cyclase (EC50 of approximately 700 nM versus 25 nM). Both the beta5 gamma2HF and the beta1 gamma2HF dimers supported coupling of M1 muscarinic receptors to the Gq alpha subunit. The ETB receptor coupled effectively to both the Gi and Gq alpha subunits in the presence of the beta1 gamma2HF dimer. In contrast, the beta5 gamma2HF dimer only supported coupling of the Gq alpha subunits to the ETB receptor and did not support coupling of the Gi alpha subunit. These results suggest that the beta5 gamma2HF dimer binds selectively to Gq alpha subunits and does not activate the same set of effectors as dimers containing the beta1 subunit. Overall, the data support a specialized role for the beta5 subunit in cell signaling.     

Selective reconstitution of gastrin-releasing peptide receptor with G alpha q

Identification of the molecular mechanisms that determine specificity of coupling interactions between gastrin-releasing peptide receptors (GRPrs) and their cognate heterotrimeric GTP-binding proteins is a fundamental step in understanding the signal transduction cascade initiated by receptor-ligand interaction. To explore these mechanisms in greater detail, we have developed an in situ reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTP gamma S to purified G protein alpha subunits. Binding studies with 125I-labeled [D-Tyr6]bombesin(6-13) methyl ester (125I-Tyr-ME), a GRPr specific antagonist, show a single binding site with a Kd = 1.4 nM +/- 0.4 (mean +/- SD, n = 3) and capacity of 15-22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2- to 3-fold enrichment of binding sites compared with the membranes before extraction. Quantitative ligand displacement analysis using various unlabeled GRPr agonists shows a rank order of potency characteristic of the GRPr: bombesin > or = GRP > > neuromedin B. Reconstitution of urea extracted membranes with a purified G alpha q showed that receptor-catalyzed binding of GTP gamma S was dependent on agonist (GRP) and G beta gamma subunits. The EC50 for GRP was 3.5 nM, which correlates well with the reported Kd of 3.1 nM for GRP binding to GRPr expressed in mouse fibroblasts [Benya, R. V., et al. (1994) Mol. Pharmacol. 46, 235-245]. The apparent Kd for bovine brain G beta gamma in this assay was 60 nM, and the Km for squid retinal G alpha q was 90 nM. The GRPr-catalyzed binding of GTP gamma S is selective for G alpha q, since we did not detect receptor-catalyzed exchange using either G alpha i/o or G alpha t. These data demonstrate that GRPr can functionally couple to G alpha q but not to the pertussis toxin-sensitive G alpha i/o or retinal specific G alpha t. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.     

Identification of sulfhydryl-modified cysteine residues in the ligand binding pocket of retinoic acid receptor beta

The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss of binding, we created three site-specific RARbeta mutants: C228A, C258A, and C267A. The affinity for RA of all three mutant receptors was in the range of that of the wild type protein, suggesting that none of these Cys residues are critical for RA binding. Rather, these modified Cys residue(s) function to sterically hinder RA binding; however, the modified Cys residues critical for the inhibition of binding differ depending on the reagent employed. Only modification of Cys228 is necessary to inhibit RA binding when RARbeta is modified by reagents which transfer large bulky groups while both Cys228 and Cys267 must be modified when a small functional group is transferred. These data suggest that both Cys228 and Cys267 but not Cys258 lie in the ligand binding pocket of RARbeta. However, Cys228 lies closer to the opening of the RARbeta ligand binding pocket whereas Cys267 lies more deeply buried.     

Metabolism to a response pathway selective retinoid ligand during axial pattern formation

We report identification of 9-cis-4-oxo-retinoic acid (9-cis-4-oxo-RA) as an in vivo retinoid metabolite in Xenopus embryos. 9-Cis-4-oxo-RA bound receptors (RARs) alpha, beta, and gamma as well as retinoid X receptors (RXRs) alpha, beta, and gamma in vitro. However, this retinoid displayed differential RXR activation depending on the response pathway used. Although it failed to activate RXRs in RXR homodimers, it activated RXRs and RARs synergistically in RAR-RXR heterodimers. 9-Cis-4-oxo-RA thus acted as a dimer-specific agonist. Considering that RAR-RXR heterodimers are major functional units involved in transducing retinoid signals during embryogenesis and that 9-cis-4-oxo-RA displayed high potency for modulating axial pattern formation in Xenopus, metabolism to 9-cis-4-oxo-RA may provide a mechanism to target retinoid action to this and other RAR-RXR heterodimer-mediated processes.     

Retinoic acid differentially regulates retinoic acid receptor-mediated pathways in the Hep3B cell line

Retinoic acid (RA) up-regulates retinoic acid receptor beta (RAR beta) gene expression in a variety of cell lines. Whether up-regulation of the RAR beta gene reflects increased activity in a RAR beta-mediated biological process is unclear since RAR beta tends to heterodimerize with retinoid x receptor (RXR). In F9 teratocarcinoma cell line, RA-induced differentiation is accompanied by increased expression of the RAR beta, RXR alpha, and alpha-fetoprotein (AFP) genes. Previously, we have shown that the RA-mediated regulation of the AFP gene is through RXR alpha homodimers. In contrast to F9 cells, Hep3B is unique in that the AFP gene is down-regulated by RA in a manner reminiscent of down-regulation of AFP in postfetal liver. In this paper, we have examined the RA-mediated regulation of the RAR, RXR, peroxisome proliferator-activated receptor (PPAR), and AFP genes in Hep3B cells. RA induced the expression of RAR alpha, beta, and gamma mRNA in Hep3B cells. However, the expression of RXR alpha mRNA was down-regulated, and the levels of RXR beta and RXR gamma mRNA remained unchanged after RA treatment. In addition, the expression of the PPAR alpha, beta, and gamma genes was also unchanged. Gel retardation assays demonstrated that RA decreased the overall binding of nuclear receptors to the RA and PPAR response elements. By super-shift assays using specific anti-RAR and -RXR antibodies, RA treatment decreased the amount of RXR alpha while increasing the amount RAR beta bound to retinoic acid response element-DR1 (direct repeat with spacer of one nucleotide), indicating the levels of RAR/RXR heterodimer, RXR/RXR homodimer, or RAR/RAR homodimers were altered upon RA treatment of Hep3B cells. In addition, the RA-mediated reduction of RXR alpha in part results in down-regulation of the AFP gene. Our data indicates that RA exerts its effects by differentially regulating its own receptor gene expression.     

A selective retinoid with high activity against an androgen-resistant prostate cancer cell type

Retinoic acid (RA) and its natural and synthetic analogs, the retinoids, regulate many biological processes, including development, differentiation, cell growth, morphogenesis, metabolism and homeostasis. Retinoid effects are mediated by specific nuclear receptors, the RARs and RXRs. Because of their ability to control cell growth and induce differentiation, retinoids are being examined for the prevention and treatment of several cancers. The majority of retinoids so far analyzed and available inhibit primarily cell proliferation and tumor progression but cannot eliminate cancer cells. In addition, the beneficial effects of the natural retinoids are undermined by undesirable side effects, possibly due to indiscriminate activation of all retinoid receptor subtypes and response pathways. Here, we show that a synthetic retinoid, CD-271, that activates selectively the RAR gamma subtype in a given context, shows increased anti-proliferative activity against certain carcinoma cells over all-trans-retinoic acid (tRA). CD-271 exhibits enhanced activity against DU-145 prostate adenocarcinoma cells through apoptosis-inducing activity, while tRA does not. The selective anti-cancer cell action appears to be receptor-mediated as an RAR antagonist reverses the inhibition. This profile was not seen with other selective retinoids, such as RAR alpha-selective agonists, anti-AP-1 compounds and a non-apoptosis inducing RAR gamma agonist. Our data point to a specific role for RAR gamma in controlling the growth of the prostate, consistent with previous RAR gamma gene knockout data. The identified retinoid represents a new class of compounds with potential for the treatment of prostate cancer.     

Beta PDGFR-IgG chimera demonstrates that human beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding

To localize human beta PDGFR binding determinants, we constructed a fusion protein comprising beta PDGFR Ig-like domains 1 to 3 and an IgG1 Fc domain (beta PDGFR-HFc). beta PDGFR-HFc was expressed as a 200 kDa dimeric molecule and contained Fc epitopes as demonstrated by anti-mouse Fc antibody recognition. Scatchard analysis revealed that PDGF BB possessed a dissociation constant of 1.5 nM for beta PDGFR-HFc. Thus, beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding. We exploited this fusion protein technology to identify and characterize beta PDGFR antagonists using a sensitive beta PDGFR immunosorbent assay. In this assay, beta PDGFR-HFc half-maximally bound to PDGF BB with an affinity of around 150 pM. Suramin, as well as bacterially expressed and refolded human alpha PDGFR domains 1-3, inhibited beta PDGFR-HFc binding to PDGF BB half-maximally at 25 microM and 10 nM respectively. Therefore, alpha PDGFR D1-3, like beta PDGFR D1-3, are sufficient for high affinity PDGF BB binding. Furthermore, the beta PDGFR-HFc immunosorbent assay will be useful to identify beta PDGFR antagonists as well as to study alpha and beta PDGFR substitution mutants which further map receptor binding determinants.     

The retinoid ligand 4-oxo-retinoic acid is a highly active modulator of positional specification

Retinoids (vitamin A and its metabolites) are suspected of regulating diverse aspects of growth, differentiation, and patterning during embryogenesis, but many questions remain about the identities and functions of the endogenous active retinoids involved. The pleiotropic effects of retinoids may be explained by the existence of complex signal transduction pathways involving diverse nuclear receptors of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families, and at least two types of cellular retinoic acid binding proteins (CRABP-I and -II). The different RARs, RXRs, and CRABPs have different expression patterns during vertebrate embryogenesis, suggesting that they each have particular functions. Another level at which fine tuning of retinoid action could occur is the metabolism of vitamin A to active metabolites, which may include all-trans-retinoic acid, all-trans-3,4-didehydroretinoic acid, 9-cis-retinoic acid, and 14-hydroxy-4,14-retroretinol. Formation of the metabolite all-trans-4-oxo-retinoic acid from retinoic acid was considered to be an inactivation pathway during growth and differentiation. We report here that, in contrast, 4-oxo-retinoic acid is a highly active metabolite which can modulate positional specification in early embryos. We also show that this retinoid binds avidly to and activates RAR beta, and that it is available in early embryos. The different activities of 4-oxo-retinoic acid and retinoic acid in modulating positional specification on the one hand, and growth and differentiation on the other, interest us in the possibility that specific retinoid ligands regulate different physiological processes in vivo.