Stella C. Martin

Molecular Identification of the Human GABABR2: Cell Surface Expression and Coupling to Adenylyl Cyclase in the Absence of GABABR1

We have identified a gene encoding a GABAB receptor, the human GABABR2, located on chromosome 9q22.1, that is distinct from the recently reported rat GABABR1. GABABR2 structurally resembles GABABR1 (35% identity), having seven transmembrane domains and a large extracellular region, but differs in having a longer carboxy-terminal tail. GABABR2 is localized to the cell surface in transfected COS cells, and negatively couples to adenylyl cyclase in response to GABA, baclofen, and 3-aminopropyl(methyl)phosphinic acid in CHO cells lacking GABABR1. Baclofen action is inhibited by the GABABR antagonist, 2-hydroxysaclofen. The human GABABR2 and GABABR1 genes are differentially expressed in the nervous system, with the greatest difference being detected in the striatum in which GABABR1 but not GABABR2 mRNA transcripts are detected. GABABR2 and GABABR1 mRNAs are also coexpressed in various brain regions such as the Purkinje cell layer of the cerebellum. Identification of a functional homomeric GABABR2 coupled to adenylyl cyclase suggests that the complexity of GABAB pharmacological data is at least in part due to the presence of more than one receptor and opens avenues for future research leading to an understanding of metabotropic GABA receptor signal transduction mechanisms.

Human GABABR genomic structure: evidence for splice variants in GABABR1 but not GABABR2

The type B gamma-aminobutryic acid receptor (GABA(B)R) is a G protein coupled receptor that mediates slow pre- and post-synaptic inhibition in the nervous system. We find that the human GABA(B)R2 gene spans greater than 350 kb and contains 2.8 kb of coding region in 19 exons. The overall similarity in genomic structure with regard to conservation of intron position and exon size between human or Drosophila GABA(B)R1 and GABA(B)R2 genes suggests a common ancestral origin. Multiple transcripts GABA(B)R1a-c and GABA(B)R2a-c have been described and alternative splicing has been proposed to result in GABA(B)R1c, GABA(B)R2b and GABA(B)R2c. The results described here provide support for the existence of GABA(B)R1c but not for GABA(B)R2b and GABA(B)R2c. Splice junctions present in the GABA(B)R1 gene sequence are consistent with the formation of GABA(B)R1c by exon skipping of one sushi domain module. The GABA(B)R2 gene lacks canonical splice junctions for the reported variants. Consistent with this, RNA analysis demonstrates the presence of GABA(B)R1c and GABA(B)R2 transcripts in fetal and adult human brain RNA but GABA(B)R2b and GABA(B)R2c transcripts are not detected. These results provide insight into the evolution and transcript diversity of the mammalian GABA(B)R genes.

The Neuroactive Steroid Pregnenolone Sulfate Stimulates Trafficking of Functional N-Methyl D-Aspartate Receptors to the Cell Surface via a Noncanonical, G Protein, and Ca2+-Dependent Mechanism

N-methyl D-aspartate (NMDA) receptors (NMDARs) mediate fast excitatory synaptic transmission and play a critical role in synaptic plasticity associated with learning and memory. NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, and clinical studies have revealed reduced negative symptoms of schizophrenia with a dose of pregnenolone that elevates serum levels of the neuroactive steroid pregnenolone sulfate (PregS). This report describes a novel process of delayed-onset potentiation whereby PregS approximately doubles the cell’s response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS. The number of functional cell-surface NMDARs in cortical neurons increases 60–100% within 10 minutes of exposure to PregS, as shown by surface biotinylation and affinity purification. Delayed-onset potentiation is reversible and selective for expressed receptors containing the NMDAR subunit subtype 2A (NR2A) or NR2B, but not the NR2C or NR2D, subunits. Moreover, substitution of NR2B J/K helices and M4 domain with the corresponding region of NR2D ablates rapid allosteric potentiation of the NMDA response by PregS but not delayed-onset potentiation. This demonstrates that the initial phase of rapid positive allosteric modulation is not a first step in NMDAR upregulation. Delayed-onset potentiation by PregS occurs via a noncanonical, pertussis toxin–sensitive, G protein–coupled, and Ca2+-dependent mechanism that is independent of NMDAR ion channel activation. Further investigation into the sequelae for PregS-stimulated trafficking of NMDARs to the neuronal cell surface may uncover a new target for the pharmacological treatment of disorders in which NMDAR hypofunction has been implicated.

Differential Expression of -Aminobutyric Acid Type B Receptor Subunit mRNAs in the Developing Nervous System and Receptor Coupling to Adenylyl Cyclase in Embryonic Neurons

γ‐Aminobutyric acid type B receptors (GABABRs) mediate both slow inhibitory synaptic activity in the adult nervous system and motility signals for migrating embryonic cortical cells. Previous papers have described the expression of GABABRs in the adult brain, but the expression and functional significance of these gene products in the embryo are largely unknown. Here we examine GABABR expression from rat embryonic day 10 (E10) to E18 compared with adult and ask whether embryonic cortical neurons contain functional GABABR. GABABR1 transcript levels greatly exceed GABABR2 levels in the developing neural tube at E11, and olfactory bulb and striatum at E17 but equalize in most regions of adult nervous tissue, except for the glomerular and granule cell layers of the main olfactory bulb and the striatum. Consistent with expression differences, the binding affinity of GABA for GABABRs is significantly lower in adult striatum compared with cerebellum. Multiple lines of evidence from in situ hybridization, RNase protection, and real‐time PCR demonstrate that GABABR1a, GABABR1b, GABABR1h (a subunit subtype, lacking a sushi domain, that we have identified in embryonic rat brain), GABABR2, and GABABL transcript levels are not coordinately regulated. Despite the functional requirement for a heterodimer of GABABR subunits, the expression of each subunit mRNA is under independent control during embryonic development, and, by E18, GABABRs are negatively coupled to adenylyl cyclase in neocortical neurons. The presence of embryonic GABABR transcripts and protein and functional receptor coupling indicates potentially important roles for GABABRs in modulation of synaptic transmission in the developing embryonic nervous system. J. Comp. Neurol. 473:16–29, 2004.

The development of neurotrophin receptor Trk immunoreactivity in the retina of the zebrafish (Brachydanio rerio)

The purpose of this study was to examine the cellular distribution of the Trk family of neurotrophin receptors in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Semithin sections from zebrafish retinae were examined immunohistochemically for the presence of Trk polypeptides using commercially available antisera that cross-react with the fish. Cross-reactivity was confirmed by Western blot. Trk polypeptides were detected at about 1 day of age on the surfaces of retinal neuroblasts and faint Trk immunoreactivity was observed in the primordial optic nerve at 1.5 days. By 2 days the optic nerve was clearly positive for Trk and at 2.5 days Trk immunoreactivity was found in the outer plexiform, inner nuclear, inner plexiform and ganglion cell layers, as well as in the optic nerve. At 3 days and 4 days the location of Trk immunoreactivity was unchanged but by 4 days it had diminished in intensity. In the adult zebrafish retina Trk immunoreactivity was found in the same locations as in the embryonic fish, as well as in a population of cells in the middle of the inner nuclear layer and in photoreceptors. We conclude that Trk neurotrophin receptors are present in the zebrafish eye during development and that their persistence in the adult may support the continuous neural reorganization that accompanies the growth of the eye in the fish.

A Role for Picomolar Concentrations of Pregnenolone Sulfate in Synaptic Activity-Dependent Ca2+ Signaling and CREB Activation

Fast excitatory synaptic transmission that is contingent upon N-methyl d-aspartate receptor (NMDAR) function contributes to core information flow in the central nervous system and to the plasticity of neural circuits that underlie cognition. Hypoactivity of excitatory NMDAR-mediated neurotransmission is hypothesized to underlie the pathophysiology of schizophrenia, including the associated cognitive deficits. The neurosteroid pregnenolone (PREG) and its metabolites pregnenolone sulfate (PregS) and allopregnanolone in serum are inversely associated with cognitive improvements after oral PREG therapy, raising the possibility that brain neurosteroid levels may be modulated therapeutically. PregS is derived from PREG, the precursor of all neurosteroids, via a single sulfation step and is present at low nanomolar concentrations in the central nervous system. PregS, but not PREG, augments long-term potentiation and cognitive performance in animal models of learning and memory. In this report, we communicate the first observation that PregS, but not PREG, is a potent (EC50 ∼2 pM) enhancer of intracellular Ca2+ that is contingent upon neuronal activity, NMDAR-mediated synaptic activity, and L-type Ca2+ channel activity. Low picomolar PregS similarly activates cAMP response element-binding protein (CREB) phosphorylation (within 10 minutes), an essential memory molecule, via an extracellular-signal-regulated kinase/mitogen-activated protein kinase signal transduction pathway. Taken together, the results are consistent with a novel biologic role for the neurosteroid PregS that acts at picomolar concentrations to intensify the intracellular response to glutamatergic signaling at synaptic but not extrasynaptic, NMDARs by differentially augmenting CREB activation. This provides a genomic signal transduction mechanism by which PregS could participate in memory consolidation of relevance to cognitive function.

Polycomblike protein PHF1b: a transcriptional sensor for GABA receptor activity

BackgroundThe γ-aminobutyric acid (GABA) type A receptor (GABAAR) contains the recognition sites for a variety of agents used in the treatment of brain disorders, including anxiety and epilepsy. A better understanding of how receptor expression is regulated in individual neurons may provide novel opportunities for therapeutic intervention. Towards this goal we have studied transcription of a GABAAR subunit gene (GABRB1) whose activity is autologously regulated by GABA via a 10 base pair initiator-like element (β1-INR).MethodsBy screening a human cDNA brain library with a yeast one-hybrid assay, the Polycomblike (PCL) gene product PHD finger protein transcript b (PHF1b) was identified as a β1-INR associated protein. Promoter/reporter assays in primary rat cortical cells demonstrate that PHF1b is an activator at GABRB1, and chromatin immunoprecipitation assays reveal that presence of PHF1 at endogenous Gabrb1 is regulated by GABAAR activation.ResultsPCL is a member of the Polycomb group required for correct spatial expression of homeotic genes in Drosophila. We now show that PHF1b recognition of β1-INR is dependent on a plant homeodomain, an adjacent helix-loop-helix, and short glycine rich motif. In neurons, it co-immunoprecipitates with SUZ12, a key component of the Polycomb Repressive Complex 2 (PRC2) that regulates a number of important cellular processes, including gene silencing via histone H3 lysine 27 trimethylation (H3K27me3).ConclusionsThe observation that chronic exposure to GABA reduces PHF1 binding and H3K27 monomethylation, which is associated with transcriptional activation, strongly suggests that PHF1b may be a molecular transducer of GABAAR function and thus GABA-mediated neurotransmission in the central nervous system.

Mechanisms of GABAA and GABAB Receptor Gene Regulation and Cell Surface Expression

The γ-aminobutyric acid (GABA) neurotransmitter acting through ionotropic and metabotropic receptor classes exerts the major inhibitory control in the central nervous system. Therapeutic agents targeting GABA receptors (GABA-R), such as benzodiazepines and baclofen, are used to treat many nervous system conditions, including anxiety and spasticity. The subunit composition of GABA-Rs at the cell surface plays a critical role in determining their physiological and pharmacological properties, and alteration of GABA-R subunit expression has been associated with a number of diseases including schizophrenia, temporal lobe epilepsy, and alcoholism. The ionotropic type A GABA receptor (GABAAR) and type C GABA receptor (GABACR) are pentameric complexes that comprise a ligand gated chloride channel. The metabotropic type B GABA receptor (GABABR) is a heterodimer that couples G protein-signaling to GABA binding. There are eight classes of ionotropic receptor subunits and only two metabotropic receptor subunit classes. Most of the GABAAR subunit genes are localized in syntenic β-α-γ gene clusters on four chromosomes but the two GABABR genes are localized on distinct chromosomes. Control over subunit expression in different brain regions and during development is orchestrated at the genomic level by the use of multiple promoter regions and through the alternative splicing of GABA-R subunit RNAs. This chapter examines current GABA-R research relevant to the many levels of control over receptor gene regulation and cell surface receptor expression that may be relevant to both health and disease.