Remodeling of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor subunit composition in hippocampal neurons after global ischemia

Transient global ischemia induces selective delayed cell death, primarily of principal neurons in the hippocampal CA1. However, the molecular mechanisms underlying ischemia-induced cell death are as yet unclear. The present study shows that global ischemia triggers a pronounced and cell-specific reduction in GluR2 [the subunit that limits Ca2+ permeability of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors] in vulnerable CA1 neurons, as evidenced by immunofluorescence of brain sections and Western blot analysis of microdissected hippocampal subfields. At 72 h after ischemia (a time before cell death), virtually all CA1 pyramidal neurons exhibited greatly reduced GluR2 immunolabeling throughout their somata and dendritic processes. GluR2 immunolabeling was unchanged in pyramidal cells of the CA3 and granule cells of the dentate gyrus, regions resistant to ischemia-induced damage. Immunolabeling of the AMPA receptor subunit GluR1 was unchanged in CA1, CA3, and dentate gyrus. Western analysis indicated that GluR2 subunit abundance was markedly reduced in CA1 at 60 and 72 h after the ischemic insult; GluR1 abundance was unchanged in all subfields at all times examined. These findings, together with the previous observation of enhanced AMPA-elicited Ca2+ influx in postischemic CA1 neurons, show that functional GluR2-lacking, Ca2+-permeable AMPA receptors are expressed in vulnerable neurons before cell death. Thus, the present study provides an important link in the postulated causal chain between global ischemia and delayed death of CA1 pyramidal neurons.

Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains

The 2.8-Å crystal structure of the complex formed by estradiol and the human estrogen receptor-α ligand binding domain (hERαLBD) is described and compared with the recently reported structure of the progesterone complex of the human progesterone receptor ligand binding domain, as well as with similar structures of steroid/nuclear receptor LBDs solved elsewhere. The hormone-bound hERαLBD forms a distinctly different and probably more physiologically important dimer interface than its progesterone counterpart. A comparison of the specificity determinants of hormone binding reveals a common structural theme of mutually supported van der Waals and hydrogen-bonded interactions involving highly conserved residues. The previously suggested mechanism by which the estrogen receptor distinguishes estradiol’s unique 3-hydroxy group from the 3-keto function of most other steroids is now described in atomic detail. Mapping of mutagenesis results points to a coactivator-binding surface that includes the region around the “signature sequence” as well as helix 12, where the ligand-dependent conformation of the activation function 2 core is similar in all previously solved steroid/nuclear receptor LBDs. A peculiar crystal packing event displaces helix 12 in the hERαLBD reported here, suggesting a higher degree of dynamic variability than expected for this critical substructure.

Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human Huntington disease gene

Loss of neurotransmitter receptors, especially glutamate and dopamine receptors, is one of the pathologic hallmarks of brains of patients with Huntington disease (HD). Transgenic mice that express exon 1 of an abnormal human HD gene (line R6/2) develop neurologic symptoms at 9–11 weeks of age through an unknown mechanism. Analysis of glutamate receptors (GluRs) in symptomatic 12-week-old R6/2 mice revealed decreases compared with age-matched littermate controls in the type 1 metabotropic GluR (mGluR1), mGluR2, mGluR3, but not the mGluR5 subtype of G protein-linked mGluR, as determined by [3H]glutamate receptor binding, protein immunoblotting, and in situ hybridization. Ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors were also decreased, while N-methyl-d-aspartic acid receptors were not different compared with controls. Other neurotransmitter receptors known to be affected in HD were also decreased in R6/2 mice, including dopamine and muscarinic cholinergic, but not γ-aminobutyric acid receptors. D1-like and D2-like dopamine receptor binding was drastically reduced to one-third of control in the brains of 8- and 12-week-old R6/2 mice. In situ hybridization indicated that mGluR and D1 dopamine receptor mRNA were altered as early as 4 weeks of age, long prior to the onset of clinical symptoms. Thus, altered expression of neurotransmitter receptors precedes clinical symptoms in R6/2 mice and may contribute to subsequent pathology.

Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: Evidence for an alcohol receptor?

Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a “cutoff” is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the α subunit. We now demonstrate that these residues in the glycine α1 and the γ-aminobutyric acid ρ1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine α1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the γ-aminobutyric acid ρ1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

Molecular evolution of two vertebrate aryl hydrocarbon (dioxin) receptors (AHR1 and AHR2) and the PAS family

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor through which halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cause altered gene expression and toxicity. The AHR belongs to the basic helix–loop–helix/Per-ARNT-Sim (bHLH-PAS) family of transcriptional regulatory proteins, whose members play key roles in development, circadian rhythmicity, and environmental homeostasis; however, the normal cellular function of the AHR is not yet known. As part of a phylogenetic approach to understanding the function and evolutionary origin of the AHR, we sequenced the PAS homology domain of AHRs from several species of early vertebrates and performed phylogenetic analyses of these AHR amino acid sequences in relation to mammalian AHRs and 24 other members of the PAS family. AHR sequences were identified in a teleost (the killifish Fundulus heteroclitus), two elasmobranch species (the skate Raja erinacea and the dogfish Mustelus canis), and a jawless fish (the lamprey Petromyzon marinus). Two putative AHR genes, designated AHR1 and AHR2, were found both in Fundulus and Mustelus. Phylogenetic analyses indicate that the AHR2 genes in these two species are orthologous, suggesting that an AHR gene duplication occurred early in vertebrate evolution and that multiple AHR genes may be present in other vertebrates. Database searches and phylogenetic analyses identified four putative PAS proteins in the nematode Caenorhabditis elegans, including possible AHR and ARNT homologs. Phylogenetic analysis of the PAS gene family reveals distinct clades containing both invertebrate and vertebrate PAS family members; the latter include paralogous sequences that we propose have arisen by gene duplication early in vertebrate evolution. Overall, our analyses indicate that the AHR is a phylogenetically ancient protein present in all living vertebrate groups (with a possible invertebrate homolog), thus providing an evolutionary perspective to the study of dioxin toxicity and AHR function.

Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix down-regulates the mRNA expression of pituitary receptors for LH-RH by counteracting the stimulatory effect of endogenous LH-RH

The mechanisms through which LH-RH antagonists suppress gonadotroph functions and LH-RH receptor (LH-RH-R) production are incompletely understood. To elucidate these mechanisms, we investigated the effects of Cetrorelix on the mRNA expression of pituitary LH-RH-R and luteinizing hormone (LH) secretion in three experimental systems with different pituitary LH-RH environments. Ovariectomy induced 3.61-fold and 6.34-fold increases in the mRNA expression of pituitary LH-RH-R in rats after 11 and 21 days, respectively. After (5 h) a single injection of 100 μg Cetrorelix, no significant decrease occurred in the mRNA levels of pituitary LH-RH-R in ovariectomized (OVX) rats with high pituitary exposure to LH-RH, but there was a significant 23.2% reduction in cycling rats with normal hypophysial LH-RH environment. Prolonged treatment for 10 days with a Cetrorelix depot formulation releasing 100 μg/day decreased the concentration of mRNA for pituitary LH-RH-R by 72.6% in OVX rats, but only by 32.9% in normal rats. The decline in serum LH was 98.7% in OVX rats and 63.2% in normal rats, resulting in a minimal 0.1–0.2 ng/ml LH concentration in both groups. A continuous exposure of pituitary cells to 100 nM Cetrorelix in the superfusion system, which is devoid of LH-RH, did not cause any significant changes in LH-RH-R mRNA level. These studies demonstrate that prolonged exposure to Cetrorelix in vivo, but not in vitro, down-regulates the mRNA expression of the pituitary receptors for LH-RH. Our findings indicate that LH-RH antagonists exert their inhibitory effects on the gene expression of pituitary LH-RH-R by counteracting the stimulatory effect of endogenous LH-RH.

Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism

Hepatotropism is a prominent feature of hepatitis B virus (HBV) infection. Cell lines of nonhepatic origin do not independently support HBV replication. Here, we show that the nuclear hormone receptors, hepatocyte nuclear factor 4 and retinoid X receptor α plus peroxisome proliferator-activated receptor α, support HBV replication in nonhepatic cells by controlling pregenomic RNA synthesis, indicating these liver-enriched transcription factors control a unique molecular switch restricting viral tropism. In contrast, hepatocyte nuclear factor 3 antagonizes nuclear hormone receptor-mediated viral replication, demonstrating distinct regulatory roles for these liver-enriched transcription factors.

Characteristics of glycine receptors expressed by embryonic rat brain mRNAs

A study was made of glycine (Gly) and γ-aminobutyric acid (GABA) receptors expressed in Xenopus oocytes injected with rat mRNAs isolated from the encephalon, midbrain, and brainstem of 18-day-old rat embryos. In oocytes injected with encephalon, midbrain, or brainstem mRNAs, the Gly-current amplitudes (membrane current elicited by Gly; 1 mM Gly) were respectively 115 ± 35, 346 ± 28, and 389 ± 22 nA, whereas the GABA-currents (1 mM GABA) were all ≤40 nA. Moreover, the Gly-currents desensitized faster in oocytes injected with encephalon or brainstem mRNAs. The EC50 for Gly was 611 ± 77 μM for encephalon, 661 ± 28 μM for midbrain, and 506 ± 18 μM for brainstem mRNA-injected oocytes, and the corresponding Hill coefficients were all ≈2. Strychnine inhibited all of the Gly-currents, with an IC50 of 56 ± 3 nM for encephalon, 97 ± 4 nM for midbrain, and 72 ± 4 nM for brainstem mRNAs. During repetitive Gly applications, the Gly-currents were potentiated by 1.6-fold for encephalon, 2.1-fold for midbrain, and 1.3-fold for brainstem RNA-injected oocytes. Raising the extracellular Ca2+ concentration significantly increased the Gly-currents in oocytes injected with midbrain and brainstem mRNAs. Reverse transcription–PCR studies showed differences in the Gly receptor (GlyR) α-subunits expressed, whereas the β-subunit was present in all three types of mRNA. These results indicate differential expression of GlyR mRNAs in the brain areas examined, and these mRNAs lead to the expression of GlyRs that have different properties. The modulation of GlyRs by Ca2+ could play important functions during brain development.

Activation of Trk neurotrophin receptors in the absence of neurotrophins

Neurotrophins regulate neuronal cell survival and synaptic plasticity through activation of Trk receptor tyrosine kinases. Binding of neurotrophins to Trk receptors results in receptor autophosphorylation and downstream phosphorylation cascades. Here, we describe an approach to use small molecule agonists to transactivate Trk neurotrophin receptors. Activation of TrkA receptors in PC12 cells and TrkB in hippocampal neurons was observed after treatment with adenosine, a neuromodulator that acts through G protein-coupled receptors. These effects were reproduced by using the adenosine agonist CGS 21680 and were counteracted with the antagonist ZM 241385, indicating that this transactivation event by adenosine involves adenosine 2A receptors. The increase in Trk activity could be inhibited by the use of the Src family-specific inhibitor, PP1, or K252a, an inhibitor of Trk receptors. In contrast to other G protein-coupled receptor transactivation events, adenosine used Trk receptor signaling with a longer time course. Moreover, adenosine activated phosphatidylinositol 3-kinase/Akt through a Trk-dependent mechanism that resulted in increased cell survival after nerve growth factor or brain-derived neurotrophic factor withdrawal. Therefore, adenosine acting through the A2A receptors exerts a trophic effect through the engagement of Trk receptors. These results provide an explanation for neuroprotective actions of adenosine through a unique signaling mechanism and raise the possibility that small molecules may be used to elicit neurotrophic effects for the treatment of neurodegenerative diseases.

Novel Nuclear Signaling Pathway Mediates Activation of Fibroblast Growth Factor-2 Gene by Type 1 and Type 2 Angiotensin II Receptors

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1′,3′-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

γ-Aminobutyric acid type A receptors modulate cAMP-mediated long-term potentiation and long-term depression at monosynaptic CA3–CA1 synapses

cAMP induces a protein-synthesis-dependent late phase of long-term potentiation (LTP) at CA3–CA1 synapses in acute hippocampal slices. Herein we report cAMP-mediated LTP and long-term depression (LTD) at monosynaptic CA3–CA1 cell pairs in organotypic hippocampal slice cultures. After bath application of the membrane-permeable cAMP analog adenosine 3′,5′-cyclic monophosphorothioate, Sp isomer (Sp-cAMPS), synaptic transmission was enhanced for at least 2 h. Consistent with previous findings, the late phase of LTP requires activation of cAMP-dependent protein kinase A and protein synthesis. There is also an early phase of LTP induced by cAMP; the early phase depends on protein kinase A but, in contrast to the later phase, does not require protein synthesis. In addition, the cAMP-induced LTP is associated with a reduction of paired-pulse facilitation, suggesting that presynaptic modification may be involved. Furthermore, we found that Sp-cAMPS induced LTD in slices pretreated with picrotoxin, a γ-aminobutyric acid type A (GABAA) receptor antagonist. This form of LTD depends on protein synthesis and protein phosphatase(s) and is accompanied by an increased ratio of failed synaptic transmission. These results suggest that GABAA receptors can modulate the effect of cAMP on synaptic transmission and thus determine the direction of synaptic plasticity.

Activation of αvβ3-Vitronectin Binding Is a Multistage Process in which Increases in Bond Strength Are Dependent on Y747 and Y759 in the Cytoplasmic Domain of β3

Integrin receptors serve as mechanical links between the cell and its structural environment. Using αvβ3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between αvβ3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the αvβ3-vitronectin binding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the β3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of αvβ3 integrin.

Activation-enhanced αIIbβ3-Integrin–Cytoskeleton Interactions Outside of Focal Contacts Require the α-Subunit

Integrins link the cell's cytoskeleton to the extracellular matrix, as well as to receptors on other cells. These links occur not only at focal contacts but also at smaller integrin-containing protein complexes outside of focal contacts. We previously demonstrated the importance of focal contact-independent integrin–cytoskeleton interactions of β2 integrins: activation of adhesion resulted from a release of integrins from cytoskeletal constraints. To determine whether changes in integrin–cytoskeleton interactions were related to activation of the integrin, we used single particle tracking to examine focal contact-independent cytoskeletal associations of αIIbβ3-integrin, in which activation results in a large conformational change. Direct activation of αIIbβ3 by mutation did not mimic activation of lymphocytes with phorbol ester, because it enhanced integrin–cytoskeleton interactions, whereas activation of lymphocytes decreased them. Using additional integrin mutants, we found that both α- and β-cytoplasmic domains were required for these links. This suggests that 1) both β2- and β3-integrins interact with the cytoskeleton outside of focal contacts; 2) activation of a cell and activation of an integrin are distinct processes, and both can affect integrin–cytoskeleton interactions; and 3) the role of the α-subunit in integrin–cytoskeleton interactions in at least some circumstances is more direct than generally supposed.

Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs.

Integrins are major two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. We report the structure-function analysis of the cytoplasmic tail of integrin beta 3 (glycoprotein IIla) based on the cellular import of synthetic peptide analogs of this region. Among the four overlapping cell-permeable peptides, only the peptide carrying residues 747-762 of the carboxyl-terminal segment of integrin beta 3 inhibited adhesion of human erythroleukemia (HEL) cells and of human endothelial cells (ECV) 304 to immobilized fibrinogen mediated by integrin beta 3 heterodimers, alpha IIb beta 3, and alpha v beta 3, respectively. Inhibition of adhesion was integrin-specific because the cell-permeable beta 3 peptide (residues 747-762) did not inhibit adhesion of human fibroblasts mediated by integrin beta 1 heterodimers. Conversely, a cell-permeable peptide representing homologous portion of the integrin beta 1 cytoplasmic tail (residues 788-803) inhibited adhesion of human fibroblasts, whereas it was without effect on adhesion of HEL or ECV 304 cells. The cell-permeable integrin beta 3 peptide (residues 747-762) carrying a known loss-of-function mutation (Ser752Pro) responsible for the genetic disorder Glanzmann thrombasthenia Paris I did not inhibit cell adhesion of HEL or ECV 304 cells, whereas the beta 3 peptide carrying a Ser752Ala mutation was inhibitory. Although Ser752 is not essential, Tyr747 and Tyr759 form a functionally active tandem because conservative mutations Tyr747Phe or Tyr759Phe resulted in a nonfunctional cell permeable integrin beta 3 peptide. We propose that the carboxyl-terminal segment of the integrin beta 3 cytoplasmic tail spanning residues 747-762 constitutes a major intracellular cell adhesion regulatory domain (CARD) that modulates the interaction of integrin beta 3-expressing cells with immobilized fibrinogen. Import of cell-permeable peptides carrying this domain results in inhibition "from within" of the adhesive function of these integrins.

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.