Evidence of functional interaction between NuMA-RARalpha and RXRalpha in an in vivo model of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is characterized by reciprocal balanced chromosomal translocations involving retinoic acid receptor-alpha (RARalpha). RARalpha heterodimerizes with the retinoid X receptor-alpha (RXRalpha) and transcriptionally regulates myeloid differentiation in response to ATRA (all-trans retinoic acid). Several lines of evidence suggest that APL fusion proteins interact with RXRalpha. To elucidate the role of RXRalpha in APL, we conditionally knocked out RXRalpha in the hCG-NuMA-RARalpha APL mouse model. Phenotype analysis of NuMA-RARalpha+ mice demonstrated that these mice developed a myeloproliferative disease-like myeloid leukemia within 4 months of birth. While hemizygous and homozygous RXRalpha conditional knockout mice were phenotypically normal as late as 12 months of age, we observed that the leukemic phenotype in NuMA-RARalpha+ mice was dependent on the presence of functional RXRalpha. Bone marrow promyelocyte counts were significantly reduced in NuMA-RARalpha+ mice with RXRalpha knocked down. Significant differences in the accumulations of Gr-1+ and Mac-1+ cells were also seen. We further observed that genes previously identified to be cooperating events in APL were also regulated in an RXRalpha-dependent manner. We therefore propose that the APL fusion protein NuMA-RARalpha cooperates with RXRalpha in the development of leukemia in hCG-NuMA-RARalpha transgenic mice and suggest a novel role for RXRalpha in the pathogenesis of APL.

Dopaminergic and GABAergic amacrine cells are direct targets of melatonin: immunocytochemical study of mt1 melatonin receptor in guinea pig retina.

Distribution of the mt1 melatonin receptor in the guinea pig retina was immunocytochemically investigated using peptide-specific anti-mt1 receptor antibody. Western blots of the guinea pig retina showed a single band at approximately 37 kilodalton (kD) immunoreactive to the anti-mt1 antibody. The most intense immunoreactivity for the mt1 receptor was detected in the cell bodies of ganglion cells. Their dendrites and axons were also immunolabeled. Subpopulations of amacrine cells, the inner plexiform layer, and the outer plexiform layer also exhibited moderate to weak immunolabeling. The mt1-positive amacrine cells were located either at the vitreal border of the inner nuclear layer or displaced in the ganglion cell layer. Double immunolabeling using antibodies to the mt1 receptor and tyrosine hydroxylase revealed that the majority of dopaminergic amacrine cells showed mt1 immunoreactivity. Almost all the ICA type dopaminergic cells were mt1 positive while the 2CA type cells less frequently exhibited mt1 immunoreaction. By double immunolabeling for the mt1 receptor and GABA, more than 50% of the mt1-immunoreactive amacrine cells were shown to be GABAergic neurons. Approximately one-third of the GABAergic amacrine cells were immunolabeled for the mt1 receptor. The present results demonstrate expression of the mt1 receptor in diverse neuronal cell types in the guinea pig retina and provide the first evidence for the direct effect of melatonin on dopaminergic and GABAergic amacrine cells via the mt1 receptor.

Expression of mt1 melatonin receptor in rat retina: evidence for multiple cell targets for melatonin.

Melatonin is synthesized in the retina at night and acts as a local modulator within this tissue by mediating the effects of darkness. We investigated the expression and localization of the mt1 (Mel1a) melatonin receptor in rat retina in order to disclose the cellular and molecular bases of melatonin's action in the mammalian retina. Western blotting of the mt1 receptor in rat retina exhibited a single immunoreactive band of approximately 37,000 mol. wt, which corresponds to the predicted molecular size of the receptor. The mt1 receptor was immunocytochemically localized to both the inner and outer plexiform layers. During postnatal development, retina from two-week-old rats showed the highest mt1 immunoreactivity; the outer plexiform layer and horizontal cell bodies were strongly immunolabeled, with weaker labeling in the inner plexiform layer. Expression of mt1 receptor messenger RNA in the rat retina was demonstrated by reverse transcription-polymerase chain reaction and in situ hybridization. mt1 receptor transcripts were localized to ganglion cells, amacrine cells and horizontal cells. These results suggest that melatonin influences retinal physiology by acting on multiple retinal cell types, including ganglion, amacrine and horizontal cells, via the mt1 receptor expressed in their processes.

Functional differentiation of multiple dopamine D1-like receptors by NNC 01-0012.

Although members of the multiple vertebrate/mammalian dopamine D1 receptor gene family can be selectively classified on the basis of their molecular/phylogenetic, structural, and tissue distribution profiles, no subtype-specific discriminating agents have yet been identified that can functionally differentiate these receptors. To define distinct pharmacological/functional attributes of multiple D1-like receptors, we analyzed the ligand binding profiles, affinity, and functional activity of 12 novel NNC compounds at mammalian/vertebrate D1/D1A and D5/D1B, as well as vertebrate D1C/D1D, dopamine receptors transiently expressed in COS-7 cells. Of all the compounds tested, only NNC 01-0012 displayed preferential selectivity for vertebrate D1C receptors, inhibiting [3H]SCH-23390 binding with an estimated affinity (approximately 0.6 nM) 20-fold higher than either mammalian/vertebrate D1/D1A or D5/D1B receptors or the D1D receptor. Functionally, NNC 01-0012 is a potent antagonist at D1C receptors, inhibiting to basal levels dopamine (10 microM)-stimulated adenylyl cyclase activity. In contrast, NNC 01-0012 (10 microM) exhibits weak antagonist activity at D1A receptors, inhibiting only 60% of maximal cyclic AMP production by dopamine, while acting as a partial agonist at vertebrate D1B and D1D receptors, stimulating adenylyl cyclase activity by approximately 33% relative to the full agonist dopamine (10 microM), an effect that was blocked by the selective D1 receptor antagonist NNC 22-0010. These data clearly suggest that the benzazepine NNC 01-0012, despite lacking the N-methyl residue in the R3 position, is a selective and potent D1C receptor antagonist. Moreover, the differential signal transduction properties exhibited by NNC 01-0012 at these receptor subtypes provide further evidence, at least in vertebrates, for the classification of the D1C receptor as a distinct D1 receptor subtype.

The dopamine D1D receptor. Cloning and characterization of three pharmacologically distinct D1-like receptors from Gallus domesticus.

Three genomic clones encoding dopamine D1-like receptors were isolated from the avian species Gallus domesticus. Two of these genes encode proteins of 451 and 488 amino acids, which, based on deduced amino acid sequence identity and homology of exhibited pharmacological profiles, appear to be species homologs of mammalian and vertebrate D1/D1A and D5/D1B receptors, respectively. The third genomic clone, termed D1D, encodes a protein of 445 amino acids displaying a deduced amino acid sequence identity within putative transmembrane domains of 75% to mammalian D1/D1A and 77% to D5/D1B receptors with overall sequence homologies of only 49% and 46%, respectively. Membranes from COS-7 cells transfected with D1D DNA bound [3H]SCH-23390 in a saturable manner with high affinity (approximately 300 pM) and with a pharmacological profile clearly indicative of a dopamine D1-like receptor. The D1D receptor exhibited affinities for 6,7-dihydroxy-2-aminotetralin and dopamine 10-fold higher than D1/D1A receptors, characteristic of the D5/D1B receptor subfamily. In contrast, the D1D receptor bound dopaminergic agents, such as SKF-38393, apomorphine, pergolide, and lisuride, with affinities 10-fold higher than other cloned mammalian or vertebrate D1A/D1B receptor subtypes, while both clozapine and haloperidol displayed considerably lower affinity for the D1D receptor. Based on the low overall amino acid sequence identity (54%) and unique pharmacological profile, the avian dopamine D1D receptor does not appear to be a species homolog of the recently cloned vertebrate D1C receptor (Sugamori, K.S., Demchyshyn, L. L., Chung, M., and Niznik, H. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10536-10540). As with all cloned mammalian and vertebrate D1-like receptors, the D1D receptor stimulates adenylate cyclase activity in the presence of dopamine or SKF-82526. Northern blot analysis reveals the selective expression of both avian D1D and D1A receptor mRNAs only in brain with the D1B receptor more widely distributed and localized in tissues such as brain, kidney, and spleen. The isolation of four distinct vertebrate dopamine D1 receptor subtypes suggests the existence of additional mammalian D1 like receptor genes that may account for the observed pharmacological and biochemical multiplicity of dopamine D1-like receptor mediated events.