Cloning of human mineralocorticoid receptor complementary DNA: structural and functional kinship with the glucocorticoid receptor.

Low-stringency hybridization with human glucocorticoid receptor (hGR) complementary DNA was used to isolate a new gene encoding a predicted 107-kilodalton polypeptide. Expression studies demonstrate its ability to bind aldosterone with high affinity and to activate gene transcription in response to aldosterone, thus establishing its identity as the human mineralocorticoid receptor (hMR). This molecule also shows high affinity for glucocorticoids and stimulates a glucocorticoid-responsive promoter. Together the hMR and hGR provide unexpected functional diversity in which hormone-binding properties, target gene interactions, and patterns of tissue-specific expression may be used in a combinatorial fashion to achieve complex physiologic control.

Beta-adrenergic receptor kinase-2 and beta-arrestin-2 as mediators of odorant-induced desensitization.

beta-Adrenergic receptor kinase (beta ARK) and beta-arrestin function in the homologous or agonist-activated desensitization of G protein-coupled receptors. The isoforms beta ARK-2 and beta-arrestin-2 are highly enriched in and localized to the dendritic knobs and cilia of the olfactory receptor neurons where the initial events of olfactory signal transduction occur. Odorants induce a rapid and transient elevation of adenosine 3',5'-monophosphate (cAMP), which activates a nonspecific cation channel and produces membrane depolarization. Preincubation of rat olfactory cilia with antibodies raised against beta ARK-2 and beta-arrestin-2 increased the odorant-induced elevation of cAMP and attenuated desensitization. These results suggest that beta ARK-2 and beta-arrestin-2 mediate agonist-dependent desensitization in olfaction.

Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide.

As a consequence of alternative RNA processing events, a single rat gene can generate messenger RNA's (mRNA's) encoding either calcitonin or a neuropeptide referred to as alpha-type calcitonin gene-related peptide (alpha-CGRP). An mRNA product of a related gene has been identified in rat brain and thyroid encoding the protein precursor of a peptide differing from alpha-CGRP by only a single amino acid. The RNA encoding this peptide, which is referred to as beta-CGRP, appears to be the only mature transcript of the beta-CGRP gene. Hybridization histochemistry reveals a similar distribution of alpha- and beta-CGRP mRNA's, but their relative levels of expression vary in different cranial nerve nuclei. Thus beta-CGRP is a new member of a family of related genes with potential functions in regulating the transduction of sensory and motor information.

Role of beta gamma subunits of G proteins in targeting the beta-adrenergic receptor kinase to membrane-bound receptors.

The rate and extent of the agonist-dependent phosphorylation of beta 2-adrenergic receptors and rhodopsin by beta-adrenergic receptor kinase (beta ARK) are markedly enhanced on addition of G protein beta gamma subunits. With a model peptide substrate it was demonstrated that direct activation of the kinase could not account for this effect. G protein beta gamma subunits were shown to interact directly with the COOH-terminal region of beta ARK, and formation of this beta ARK-beta gamma complex resulted in receptor-facilitated membrane localization of the enzyme. The beta gamma subunits of transducin were less effective at both enhancing the rate of receptor phosphorylation and binding to the COOH-terminus of beta ARK, suggesting that the enzyme preferentially binds specific beta gamma complexes. The beta gamma-mediated membrane localization of beta ARK serves to intimately link receptor activation to beta ARK-mediated desensitization.

Kinetics of a human glutamate transporter.

Currents mediated by a glutamate transporter cloned from human motor cortex were measured in Xenopus oocytes. In the absence of glutamate, voltage jumps induced Na(+)-dependent capacitive currents that were blocked by kainate, a competitive transport antagonist. The pre-steady-state currents can be described by an ordered binding model in which a voltage-dependent Na+ binding is followed by a voltage-independent kainate binding. At -80 mV, two charges are translocated per molecule of glutamate, with a cycling time of approximately 70 ms, which is significantly slower than the predicted time course of synaptically released glutamate. The results suggest that glutamate diffusion and binding to transporters, rather than uptake, are likely to dominate the synaptic concentration decay kinetics.

The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response.

The cloning of the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) cDNAs provides a basis for understanding the actions of glucocorticoids in the central nervous system. Structural evidence is presented for the identity of the type I corticosteroid binding site as the MR expressed in the brain. This identification is supported by the anatomical distribution of MR mRNA, determined by in situ hybridization histochemistry, which parallels the steroid autoradiographic localization of the type I sites. An in vitro assay for MR and GR function demonstrates that these receptors respond to different levels of glucocorticoid, suggesting that together they confer a larger dynamic range of sensitivity to this hormone. These studies lead to a new hypothesis for glucocorticoid action in the central nervous system.

Neurotransmitter transporters: three distinct gene families.

Our understanding of the plasma membrane and vesicular transport systems that mediate neurotransmitter re-uptake has been greatly enhanced in the past year by the cloning and characterization of two additional gene families involved in this process, the excitatory amino acid transporters and the vesicular amine transporters. Additional members of the previously defined family of Na+/Cl(-)-dependent transporters continue to be identified.

Transactivation and synergistic properties of the mineralocorticoid receptor: relationship to the glucocorticoid receptor.

The human mineralocorticoid (hMR) and glucocorticoid (hGR) receptors mediate biological responses to adrenal corticosteroids and synthetic ligands. In transient transfection studies, corticosteroid-responsive promoters were used to monitor the hormone-dependent transcriptional regulatory properties of both receptors. The hMR mediates a lower stimulation of the transcription rate than the hGR and does not show cooperative activity on promoters containing multiple palindromic glucocorticoid-responsive elements. The functional importance of the amino-terminus in this differential response was demonstrated by hMR/hGR hybrid receptors in which this region was exchanged or deleted. These experiments revealed that the hMR amino-terminus does not provide the strong transactivation function present in the equivalent hGR domain and, in contrast to the hGR amino-terminus, interferes with the synergistic activity mediated by the DNA- and ligand-binding domains of both receptors.

Localization and function of five glutamate transporters cloned from the salamander retina.

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. Native glutamate transporters have been well characterized in several retinal neurons, particularly from the salamander retina. We have cloned five distinct glutamate transporters from the salamander retina and examined their localization and functional properties: sEAAT1, sEEAAT2A, sEAAT2B, sEAAT5A and sEAAT5B. sEAAT1 is a homologue of the glutamate transporter EAAT1 (GLAST), sEAAT2A and sEAAT2B are homologues of EAAT2 (GLT-1) and sEAAT5A and sEAAT5B are homologues of the recently cloned human retinal glutamate transporter EAAT5. Localization was determined by immunocytochemical techniques using antibodies directed at portions of the highly divergent carboxy terminal. Glutamate transporters were found in glial, photoreceptor, bipolar, amacrine and ganglion cells. The pharmacology and ionic dependence were determined by two-electrode voltage clamp recordings from Xenopus laevis oocytes which had previously been injected with one of the glutamate transporter mRNAs. Each of the transporters behaved in a manner consistent with a glutamate transporter and there were some distinguishing characteristics which make it possible to link the function in native cells with the behavior of the cloned transporters in this study.

Characterization of alpha 2-adrenergic receptor subtype-specific antibodies.

Subtypes of alpha 2-adrenergic receptors have been defined pharmacologically in a variety of mammalian tissues. The alpha 2A, alpha 2B, alpha 2C, and most recently alpha 2D subtypes have been characterized by their affinities for selective receptor antagonists and agonists. The genes that may encode the alpha 2A, alpha 2B, and alpha 2C subtypes have been identified in human and rat. In human these genes are termed alpha 2-C10, alpha 2-C2, and alpha 2-C4, respectively, based on their chromosomal localization, whereas three genes, designated RG20 alpha 2, RNG alpha 2, and RG10 alpha 2, are thought to be the corresponding rat homologues. These assignments were based on the pharmacology of the cloned receptor genes expressed in transfected cells and on the detection of homologous mRNAs by Northern blot analyses in cell lines or tissues with pharmacologically defined alpha 2-adrenergic receptors. However, the subtype assignment of cloned genes has not been fully resolved by these means. To help clarify the subtype assignment, we have raised antibodies against sequences from the divergent third intracellular loop of the human and rat alpha 2-adrenergic receptors. These antibodies were found to be subtype specific in immunoprecipitating either the cloned receptors expressed by DNA transfection or the pharmacologically defined receptors prepared from various tissues. Our immunological data corroborate the assignments of alpha 2-C2/RNG alpha 2 as encoding the alpha 2B subtype in NG108-15 cells and rat neonatal lung and of alpha 2-C4/RG10 alpha 2 as encoding the alpha 2C subtype in opossum kidney cells. Furthermore, antibodies against alpha 2-C10 and RG20 alpha 2 but not alpha 2-C2/RNG alpha 2 or alpha 2-C4/RG10 alpha 2 were both found to recognize alpha 2-adrenergic receptors expressed in rat submaxillary glands and in bovine pineal gland, two tissues with alpha 2D pharmacology. Because three genes were identified in the rat and human genome, these data suggest that the pharmacologically defined "alpha 2D receptor" is genetically of the alpha 2A subtype.

Differential modulation of human glutamate transporter subtypes by arachidonic acid.

Arachidonic acid has been proposed to be a messenger molecule released following synaptic activation of glutamate receptors and during ischemia. Here we demonstrate that micromolar levels of arachidonic acid inhibit glutamate uptake mediated by EAAT1, a human excitatory amino acid transporter widely expressed in brain and cerebellum, by reducing the maximal transport rate approximately 30%. In contrast, arachidonic acid increased transport mediated by EAAT2, a subtype abundantly expressed in forebrain and midbrain, by causing the apparent affinity for glutamate to increase more than 2-fold. The results demonstrate that the response of different glutamate transporter subtypes to arachidonic acid could influence synaptic transmission and modulate excitotoxicity via positive or negative feedback according to the transporter(s) present in a particular region.

Constitutive ion fluxes and substrate binding domains of human glutamate transporters.

Application of L-glutamate activates ionic currents in voltage-clamped Xenopus oocytes expressing cloned human excitatory amino acid transporters (EAATs). However, even in the absence of L-glutamate, the membrane conductance of oocytes expressing EAAT1 was significantly increased relative to oocytes expressing EAAT2 or control oocytes. Whereas transport mediated by EAAT2 is blocked by the non-transported competitive glutamate analog kainate (Ki = 14 microM), EAAT1 is relatively insensitive (Ki > 3 mM). Substitution of a block of 76 residues from EAAT2 into EAAT1, in which 18 residues varied from EAAT1, conferred high affinity kainate binding to EAAT1, and application of kainate to oocytes expressing the chimeric transporter blocked a pre-existing monovalent cation conductance that displayed a permeability sequence K+ > Na+ > Li+ >> choline+. The results identify a structural domain of glutamate transporters that influences kainate binding and demonstrate the presence of a constitutive ion-selective pore in the transporter.

Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium ions and one chloride ion.

Excitatory amino acid transporter 5, a retinal glutamate transporter coupled to a chloride conductance.

Although a glutamate-gated chloride conductance with the properties of a sodium-dependent glutamate transporter has been described in vertebrate retinal photoreceptors and bipolar cells, the molecular species underlying this conductance has not yet been identified. We now report the cloning and functional characterization of a human excitatory amino acid transporter, EAAT5, expressed primarily in retina. Although EAAT5 shares the structural homologies of the EAAT gene family, one novel feature of the EAAT5 sequence is a carboxy-terminal motif identified previously in N-methyl-D-aspartate receptors and potassium channels and shown to confer interactions with a family of synaptic proteins that promote ion channel clustering. Functional properties of EAAT5 were examined in the Xenopus oocyte expression system by measuring radiolabeled glutamate flux and two-electrode voltage clamp recording. EAAT5-mediated L-glutamate uptake is sodium- and voltage-dependent and chloride-independent. Transporter currents elicited by glutamate are also sodium- and voltage-dependent, but ion substitution experiments suggest that this current is largely carried by chloride ions. These properties of EAAT5 are similar to the glutamate-elicited chloride conductances previously described in retinal neurons, suggesting that the EAAT5-associated chloride conductance may participate in visual processing.

Regional chromosomal assignment of the human mineralocorticoid receptor gene to 4q31.1.

The gene for human mineralocorticoid receptor (hMR), previously mapped to chromosome 4, has been further localized to 4q31.1 by in situ hybridization using a biotinylated 3.75 kb human cDNA clone encoding the primary amino acid sequence of hMR as a probe. Preliminary comparative mapping studies in orangutan (Pongo pygmaeus) suggest localization of the probe to the long arm of chromosome 3.

Excitatory amino acid transporters of the salamander retina: identification, localization, and function.

The rapid re-uptake of extracellular glutamate mediated by a family of high-affinity glutamate transporter proteins is essential to continued glutamatergic signaling and neuronal viability, but the contributions of individual transporter subtypes toward cellular physiology are poorly understood. Because the physiology of glutamate transport in the salamander retina has been well described, we have examined the expression and function of glutamate transporter subtypes in this preparation. cDNAs encoding five distinct salamander excitatory amino acid transporter (sEAAT) subtypes were isolated, and their molecular properties and distributions of expression were compared. We report evidence that at least four distinct sEAAT subtypes are expressed in glial (Müller) cells. In addition, four of the five transporter subtypes are localized in neurons throughout the retina. The brightest immunostaining was seen in the synaptic regions of the inner and outer plexiform layers and in the outer nuclear layer. Using electrophysiological measurements in the Xenopus oocyte expression system, we also examined the pharmacology and ionic dependence of the four expressing transporter subtypes that make it possible to distinguish, on the basis of functional behavior, among the various subtypes. Although no simple correlation between transporter subtype and retinal cell physiology can be made, the diverse population of sEAAT transporter subtypes with unique localization and functional properties indicates that glutamate transporters play a wide variety of roles in retinal function and are likely to underlie both the uptake of glutamate by Müller cells and the glutamate-elicited chloride conductance involved in signal transduction by photoreceptors and bipolar cells.

An excitatory amino-acid transporter with properties of a ligand-gated chloride channel.

Excitatory amino-acid transporters (EAATs) in the central nervous system maintain extracellular glutamate concentrations below excitotoxic levels and may limit the activation of glutamate receptors. Here we report the cloning of a novel human aspartate/glutamate transporter, EAAT4, which is expressed predominantly in the cerebellum. The transport activity encoded by EAAT4 has high apparent affinity for L-aspartate and L-glutamate, and has a pharmacological profile consistent with previously described cerebellar transport activities. In Xenopus oocytes expressing EAAT4, L-aspartate and L-glutamate elicited a current predominantly carried by chloride ions. This chloride conductance was not blocked by components that block endogenous oocyte chloride channels. Thus EAAT4 combines the re-uptake of neurotransmitter with a mechanism for increasing chloride permeability, both of which could regulate excitatory neurotransmission.

Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex.

Reuptake plays an important role in regulating synaptic and extracellular concentrations of glutamate. Three glutamate transporters expressed in human motor cortex, termed EAAT1, EAAT2, and EAAT3 (for excitatory amino acid transporter), have been characterized by their molecular cloning and functional expression. Each EAAT subtype mRNA was found in all human brain regions analyzed. The most prominent regional variation in message content was in cerebellum where EAAT1 expression predominated. EAAT1 and EAAT3 mRNAs were also expressed in various non-nervous tissues, whereas expression of EAAT2 was largely restricted to brain. The kinetic parameters and pharmacological characteristics of transport mediated by each EAAT subtype were determined in transfected mammalian cells by radio-label uptake and in microinjected oocytes by voltage-clamp measurements. The affinities of the EAAT subtypes for L-glutamate were similar, with Km determinations varying from 48 to 97 microM in the mammalian cell assay and from 18 to 28 microM in oocytes. Glutamate uptake inhibitors were used to compare the pharmacologies of the EAAT subtypes. The EAAT2 subtype was distinguishable from the EAAT1/EAAT3 subtypes by the potency of several inhibitors, but most notably by sensitivity to kainic acid (KA) and dihydrokainic acid (DHK). KA and DHK potently inhibited EAAT2 transport, but did not significantly affect transport by EAAT1/EAAT3. Using voltage-clamp measurements, most inhibitors were found to be substrates that elicited transport currents. In contrast, KA and DHK did not evoke currents and they were found to block EAAT2-mediated transport competitively. This selective interaction with the EAAT2 subtype could be a significant factor in KA neurotoxicity. These studies provide a foundation for understanding the role of glutamate transporters in human excitatory neurotransmission and in neuropathology.

The mouse and human excitatory amino acid transporter gene (EAAT1) maps to mouse chromosome 15 and a region of syntenic homology on human chromosome 5.

The gene for human excitatory amino acid transporter (EAAT1) was localized to the distal region of human chromosome 5p13 by in situ hybridization of metaphase chromosome spreads. Interspecific back-cross analysis identified the mouse Eaat1 locus in a region of 5p13 homology on mouse chromosome 15. Markers that are linked with EAAT1 on both human and mouse chromosomes include the receptors for leukemia inhibitory factor, interleukin-7, and prolactin. The Eaat1 locus appears not to be linked to the epilepsy mutant stg locus, which is also on chromosome 15. The EAAT1 locus is located in a region of 5p deletions that have been associated with mental retardation and microcephaly.