Identification of a second human retinoic acid receptor

We have previously described a human complementary DNA that encodes a novel protein which is homologous to members of the steroid/thyroid nuclear receptor multigene family. This novel protein (hap for hepatoma) exhibits strong homology with the human retinoic acid receptor (RAR) which has been recently characterized. To test the possibility that the hap protein might also be a retinoid receptor, a chimaeric receptor was created by replacing the putative DNA binding domain of hap with that of the human oestrogen receptor (ER). The resulting hap-ER chimaera was then tested for its ability to trans-activate an oestrogen-responsive reporter gene (vit-tk-CAT) in the presence of possible receptor ligands. Here we show that retinoic acid (RA) at physiological concentrations is effective in inducing the expression of this reporter gene by the hap-ER chimaeric receptor. This demonstrates the existence of two human retinoic acid receptors designated RAR-alpha and RAR-beta.     

A new retinoic acid receptor identified from a hepatocellular carcinoma

Processes as diverse as growth, vision and reproduction depend on the presence of vitamin A and its metabolites (retinoids), but the molecular mechanisms which govern these diverse actions remain unclear (for reviews see refs 1,2). A crucial advance recently was the isolation of a specific nuclear receptor for retinoic acid, one of the physiologically active vitamin A derivatives. This nuclear receptor is a member of the steroid/thyroid hormone receptor family. Our analysis of an uncharacterized member of this class of intracellular receptors, encoded by a complementary DNA clone from a human placental library, has led us to discover a second retinoic acid receptor. This new receptor is expressed at high levels in a number of epithelial-type tissues. The gene for the receptor was first identified in a hepatocellular carcinoma where it surrounds a site of integration of hepatitis B virus. Activation by this virus may play a role in tumour development in liver cells, where it is normally not expressed.     

Spatial and temporal expression of the retinoic acid receptor in the regenerating amphibian limb

Retinoic acid is known to have dramatic effects on vertebrate limb pattern in development and regeneration, supporting a model in which a gradient of retinoic acid serves as a morphogen to differentially supply positional information to a developing limb. The discovery of a retinoic acid receptor (RAR) and its homology to the steroid and thyroid hormone receptors provided a potential molecular mechanism for limb morphogenesis. One prediction of this model is that the receptor must be expressed in the developing and regenerating limb anlage. We investigated the expression of the RAR in the adult newt, Notophthalmus viridescens, whose amputated limbs are capable of regenerating and upon which retinoic acid can act to alter pattern. We report the cloning of cDNAs encoding a functional newt RAR and the localization of high and uniform levels of RAR mRNA specifically in the regenerating cells that control limb pattern. These results indicate that the morphogenic field is established through differential activation of pre-existing retinoic acid receptors rather than differential expression of the RAR gene.     

A human retinoic acid receptor which belongs to the family of nuclear receptors

A cDNA encoding a protein that binds retinoic acid with high affinity has been cloned. The protein is homologous to the receptors for steroid hormones, thyroid hormones and vitamin D3, and appears to be a retinoic acid-inducible trans-acting enhancer factor, suggesting that the molecular mechanisms of the effect of retinoids (vitamin A) on embryonic development, differentiation and tumour cell growth are similar to those described for other members of this nuclear receptor family.     

9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.     

Homodimer formation of retinoid X receptor induced by 9-cis retinoic acid.

Retinoid response pathways are mediated by two classes of receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). A central question is whether distinct response pathways are regulated by these two classes of receptors. The observation that the stereoisomer 9-cis-retinoic acid binds with high affinity to RXRs suggested that this retinoid has a distinct role in controlling RXR activity, but it was almost simultaneously discovered that RXRs function as auxiliary receptors for RARs and related receptors, and are essential for DNA binding and function of those receptors. Hence, although RARs seem to operate effectively only as heterodimeric RAR/RXR complexes, RXRs themselves apparently function predominantly, if not exclusively, as auxiliary receptors. Here we report that 9-cis-retinoic acid induces RXR homodimer formation. Our results demonstrate a new mechanism for retinoid action by which a ligand-induced homodimer mediates a distinct retinoid response pathway.     

Identification of a ribosome receptor in the rough endoplasmic reticulum

Attachment of ribosomes to the membrane of the endoplasmic reticulum is one of the crucial first steps in the transport and secretion of intracellular proteins in mammalian cells. The process is mediated by an integral membrane protein of relative molecular mass 180,000 (Mr 180K), having a large (at least 160K) cytosolic domain that, when proteolytically detached from the membrane, can competitively inhibit the binding of ribosomes to intact membranes. Isolation of this domain has led to the identification, purification and characterization of the intact ribosome receptor, as well as its functional reconstitution into lipid vesicles.     

Identification of a putative second T-cell receptor

Framework monoclonal antibodies have identified a population of human lymphocytes that express the T3 glycoprotein but not the T-cell receptor (TCR) alpha- and beta-subunits. Chemical crosslinking experiments reveal that these lymphocytes express novel T3-associated polypeptides, one of which appears to be the product of the T gamma gene. The other polypeptide may represent a fourth TCR subunit, designated T delta.     

Evidence that Hensen's node is a site of retinoic acid synthesis.

Hensen's node of amniotes, like the Spemann organizer of amphibians, can induce a second body axis when grafted into a host embryo. The avian node, as well as several midline structures originating from it (notochord, floor plate), can also induce digit pattern duplications when grafted into the chick wing bud. We report here that the equivalent of Hensen's node from mouse is an effective inducer of digits in the chick wing bud. Tissues anterior and posterior to the node also evoke pattern duplications, but with a significantly lower efficiency. The finding that the murine node operates in an avian wing bud suggests that the same inducing agent(s) function in both primary and secondary embryonic fields and have been conserved during vertebrate evolution. Digit pattern duplications are also evoked by local administration of all-trans-retinoic acid. This similarity raises the possibility that Hensen's node is a source of retinoic acid. The mouse node is capable of synthesizing retinoic acid from its biosynthetic precursor all-trans-retinol at a substantially higher rate than either anterior or posterior tissues.     

维甲酸纳米颗粒混悬剂的制备及表征

采用溶剂分散法制备了不同药物质量浓度的维甲酸纳米颗粒混悬剂,并对其进行了冷冻干燥;采用光子相关谱(PCS)测定了冷冻干燥前后混悬剂中纳米粒子的平均粒径;透射电镜(TEM)观察其微观形貌;高效液相色谱法(HPLC)对不同时间混悬剂中全反式维甲酸的质量浓度进行定量测定．结果表明,混悬剂中维甲酸纳米粒子(RA-NP)的粒径保持在200～400nm之间,大都为完整的球形;与维甲酸硅油10～15mg／L的药物质量浓度相比,纳米颗粒混悬剂中有效药物的质量浓度可显著提高至35mg／L;药物降解速度得到明显降低,4周之内能维持抑制细胞增殖的有效药物质量浓度．维甲酸纳米颗粒混悬剂有望成为一种新型的增殖性玻璃体视网膜病变(PVR)治疗用药物载体．     

Identification of Tim4 as a phosphatidylserine receptor

In programmed cell death, a large number of cells undergo apoptosis, and are engulfed by macrophages to avoid the release of noxious materials from the dying cells. In definitive erythropoiesis, nuclei are expelled from erythroid precursor cells and are engulfed by macrophages. Phosphatidylserine is exposed on the surface of apoptotic cells and on the nuclei expelled from erythroid precursor cells; it works as an 'eat me' signal for phagocytes. Phosphatidylserine is also expressed on the surface of exosomes involved in intercellular signalling. Here we established a library of hamster monoclonal antibodies against mouse peritoneal macrophages, and found an antibody that strongly inhibited the phosphatidylserine-dependent engulfment of apoptotic cells. The antigen recognized by the antibody was identified by expression cloning as a type I transmembrane protein called Tim4 (T-cell immunoglobulin- and mucin-domain-containing molecule; also known as Timd4). Tim4 was expressed in Mac1+ cells in various mouse tissues, including spleen, lymph nodes and fetal liver. Tim4 bound apoptotic cells by recognizing phosphatidylserine via its immunoglobulin domain. The expression of Tim4 in fibroblasts enhanced their ability to engulf apoptotic cells. When the anti-Tim4 monoclonal antibody was administered into mice, the engulfment of apoptotic cells by thymic macrophages was significantly blocked, and the mice developed autoantibodies. Among the other Tim family members, Tim1, but neither Tim2 nor Tim3, specifically bound phosphatidylserine. Tim1- or Tim4-expressing Ba/F3 B cells were bound by exosomes via phosphatidylserine, and exosomes stimulated the interaction between Tim1 and Tim4. These results indicate that Tim4 and Tim1 are phosphatidylserine receptors for the engulfment of apoptotic cells, and may also be involved in intercellular signalling in which exosomes are involved.     

Identification of the haemoglobin scavenger receptor

Intravascular haemolysis is a physiological phenomenon as well as a severe pathological complication when accelerated in various autoimmune, infectious (such as malaria) and inherited (such as sickle cell disease) disorders. Haemoglobin released into plasma is captured by the acute phase protein haptoglobin, which is depleted from plasma during elevated haemolysis. Here we report the identification of the acute phase-regulated and signal-inducing macrophage protein, CD163, as a receptor that scavenges haemoglobin by mediating endocytosis of haptoglobin-haemoglobin complexes. CD163 binds only haptoglobin and haemoglobin in complex, which indicates the exposure of a receptor-binding neoepitope. The receptor-ligand interaction is Ca2+-dependent and of high affinity. Complexes of haemoglobin and multimeric haptoglobin (the 2-2 phenotype) exhibit higher functional affinity for CD 163 than do complexes of haemoglobin and dimeric haptoglobin (the 1-1 phenotype). Specific CD163-mediated endocytosis of haptoglobin-haemoglobin complexes is measurable in cells transfected with CD163 complementary DNA and in CD163-expressing myelo-monocytic lymphoma cells.