The Lac repressor provides a reversible gene expression system in undifferentiated and differentiated embryonic stem cell

Control of mammalian gene promoters by the bacterial LacI repressor provides reversible regulation and dose-response levels of derepressed expression by the lactose analog isopropyl thiogalactose (IPTG). Here, we show that insertion of LacI-binding sites in the ubiquitous β-actin promoter confers a strong and dose-dependent IPTG-regulatable expression of transiently transfected reporter genes in mouse embryonic stem (ES) cells expressing LacI. We established ES cell lines stably expressing reporter genes under inducible control and found a five- to tenfold IPTG induction of transgene expression. The kinetics of induction is rapid and stable, and can be rapidly reversed after IPTG removal. Importantly, this regulatable expression was maintained throughout the differentiation process of ES cells, and observed in individual differentiated cardiomyocyte-like cells and neuronal-like cells. This reversible system is the first to function from undifferentiated to individual welldifferentiated ES cells, providing a very useful tool to understand molecular mechanisms underlying ES cell self-renewal, commitment and differentiation.Received 17 March 2005; received after revision 19 April 2005; accepted 25 April 2005     

Cloning of the gene and cDNA for mammalian beta-adrenergic receptor and homology with rhodopsin

The adenylate cyclase system, which consists of a catalytic moiety and regulatory guanine nucleotide-binding proteins, provides the effector mechanism for the intracellular actions of many hormones and drugs. The tissue specificity of the system is determined by the particular receptors that a cell expresses. Of the many receptors known to modulate adenylate cyclase activity, the best characterized and one of the most pharmacologically important is the beta-adrenergic receptor (beta AR). The pharmacologically distinguishable subtypes of the beta-adrenergic receptor, beta 1 and beta 2 receptors, stimulate adenylate cyclase on binding specific catecholamines. Recently, the avian erythrocyte beta 1, the amphibian erythrocyte beta 2 and the mammalian lung beta 2 receptors have been purified to homogeneity and demonstrated to retain binding activity in detergent-solubilized form. Moreover, the beta-adrenergic receptor has been reconstituted with the other components of the adenylate cyclase system in vitro, thus making this hormone receptor particularly attractive for studies of the mechanism of receptor action. This situation is in contrast to that for the receptors for growth factors and insulin, where the primary biochemical effectors of receptor action are unknown. Here, we report the cloning of the gene and cDNA for the mammalian beta 2AR. Analysis of the amino-acid sequence predicted for the beta AR indicates significant amino-acid homology with bovine rhodopsin and suggests that, like rhodopsin, beta AR possesses multiple membrane-spanning regions.     

Effect of protein kinase C activation and depletion on insulin stimulation of glycogen synthesis in cultured hepatoma cells

Insulin stimulation of glycogen synthesis was nearly abolished in hepatoma cells shortly treated with 4 beta-phorbol 12 beta-myristate, 13 alpha-acetate (protein kinase C activation) but remained unmodified in cells chronically treated with the phorbol ester (protein kinase C depletion). Thus, although exogenous activation of protein kinase C results in an inhibition of insulin action, protein kinase C depletion has no influence on this process. The results suggest that, in hepatoma cells, no endogenous activation of protein kinase C may occur in response to the signal triggered by insulin.     

Rhodamine isothiocyanate coupled peanut lectin for quantitative studies of D-galactosyl receptors of neuroblastoma cells

Summary Rhodamine-peanut agglutinin conjugate has been obtained without alteration of the lectin activity. This conjugate interacts specifically with terminal D-galactosyl glycoconjugates. Such receptors are found on neuroblastoma differentiated or undifferentiated cell membranes. The density and distribution of these galactosyl sites are different after neuraminidase treatment.Acknowledgment. Part of this work was supported by a contract from the Délégation Générale à la Recherche Scientifique, n^o 77.7.1273.     

Isoprenylation in regulation of signal transduction by G-protein-coupled receptor kinases.

Rhodopsin kinase and beta-adrenergic receptor kinase (beta ARK) are related members of a serine/threonine kinase family that specifically initiate deactivation of G-protein-coupled receptors. After stimulus-mediated receptor activation, these cytoplasmic kinases translocate to the plasma membrane. Here we show that the molecular basis for this event involves a class of unsaturated lipids called isoprenoids. Covalent modification in vivo of rhodopsin kinase by a 15-C (farnesyl) isoprenoid enables the kinase to anchor to photon-activated rhodopsin. Mutations that alter or eliminate the isoprenoid, fully disable light-specific Rhodopsin kinase translocation. Other receptor kinases (such as beta ARK), which lack an intrinsic lipid, are activated on exposure to brain beta gamma subunits of the signal-transducing G proteins, the gamma subunit of which bears a 20-C (geranylgeranyl) isoprenoid. Using chimaeric beta ARKs that undergo isoprenylation in vitro, we demonstrate that membrane association and activation of these kinases can occur in the absence of beta gamma. These results indicate that rhodopsin kinase (by means of an integral isoprenoid) and beta ARK (through its association with beta gamma) both rely on the function of isoprenyl moieties for their translocation and activity, illustrating distinct, though related, modes of biological regulation of receptor function.     

Functional polymorphisms of the brain serotonin synthesizing enzyme tryptophan hydroxylase-2

Many neuropsychiatric disorders are considered to be related to the dysregulation of brain serotonergic neurotransmission. Tryptophan hydroxylase-2 (TPH2) is the neuronal-specific enzyme that controls brain serotonin synthesis. There is growing genetic evidence for the possible involvement of TPH2 in serotonin-related neuropsychiatric disorders; however, the degree of genetic variation in TPH2 and, in particular, its possible functional consequences remain unknown. In this short review, we will summarize some recent findings with respect to the functional analysis of TPH2. Received 12 September 2005; received after revision 25 October 2005; accepted 31 October 2005     

The genomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the 5-HT1A receptor

The recent cloning of the complementary DNAs and/or genes for several receptors linked to guanine nucleotide regulatory proteins including the adrenergic receptors (alpha 1, alpha 2A, alpha 2B, beta 1, beta 2), several subtypes of the muscarinic cholinergic receptors, and the visual 'receptor' rhodopsin has revealed considerable similarity in the primary structure of these proteins. In addition, all of these proteins contain seven putative transmembrane alpha-helices. We have previously described a genomic clone, G-21, isolated by cross-hybridization at reduced stringency with a full length beta 2-adrenergic receptor probe. This clone contains an intronless gene which, because of its striking sequence resemblance to the adrenergic receptors, is presumed to encode a G-protein-coupled receptor. Previous attempts to identify this putative receptor by expression studies have failed. We now report that the protein product of the genomic clone, G21, transiently expressed in monkey kidney cells has all the typical ligand-binding characteristics of the 5-hydroxytryptamine (5-HT1A) receptor.