Novel putative receptor tyrosine kinase encoded by the melanoma-inducing Tu locus in Xiphophorus

No abstract availabl

Rapid identification of recombinant baculoviruses using PCR

no abstract availabl

Subcellular localization of GABA(B) receptor subunits in rat visual cortex

Although studies in the visual cortex have found gamma-aminobutyric acid B (GABA(B)) receptor-mediated pre- and postsynaptic inhibitory effects on neurons, the subcellular localization of GABA(B) receptors in different types of cortical neurons and synapses has not been shown directly. To provide this information, we have used antibodies against the GABA(B) receptor (R)1a/b and GABA(B)R2 subunits and have studied the localization of immunoreactivities in rat visual cortex. Light microscopic analyses have shown that both subunits are expressed in cell bodies and dendrites of 65-92% of corticocortically projecting pyramidal neurons and in 92-100% of parvalbumin (PV)-, calretinin (CR)-, and somatostatin (SOM)-containing GABAergic neurons. Electron microscopic analyses of immunoperoxidase- and immunogold-labeled tissue revealed staining in the nucleus, cytoplasm and cell surface membranes with both antibodies. Colocalization of both subunits was observed in all of these structures. GABA(B)R1a/b and GABA(B)R2 were concentrated in excitatory and inhibitory synapses and in extrasynaptic membranes. In GABAergic synapses, GABA(B)R1a/b and GABA(B)R2 were more strongly expressed postsynaptically on pyramidal and nonpyramidal cells than presynaptically. In type 1 synapses GABA(B)R1a/b and GABA(B)R2 was found in pre- and postsynaptic membranes. The nuclear localization of GABA(B)R1 and GABA(B)R2 subunits suggests a novel role for neurotransmitter receptors in controlling gene expression. The synaptic colocalization of GABA(B)R1 and GABA(B)R2 indicates that subunits form heteromeric assemblies of the functional receptor in inhibitory and excitatory synapses. Subunit coexpression in GABAergic synapses that include PV-containing and PV-deficient terminals suggests that pre- and postsynaptic GABA(B) receptor activation is provided by several different types of interneurons. The coexpression of both subunits in excitatory synapses suggests a role for GABA(B)receptors in the regulation of glutamate release and raises the question how these receptors are activated in the absence of pre-or postsynaptic GABAergic synaptic inputs to excitatory synapse

The Xmrk receptor tyrosine kinase is activated in Xiphophorus malignant melanoma.

Xmrk encodes a putative transmembrane glycoprotein of the tyrosine kinase family and is a melanoma-inducing gene in Xiphophorus. We attempted to investigate the biological function of the putative Xmrk receptor by characterizing its signalling properties. Since a potential ligand for Xmrk has not yet been identified, it has been difficult to analyse the biochemical properties and biological function of this cell surface protein. In an approach towards such analyses, the Xmrk extracellular domain was replaced by the closely related ligand-binding domain sequences of the human epidermal growth factor receptor (HER) and the ligand-induced activity of the chimeric HER-Xmrk protein was examined. We show that the Xmrk protein is a functional receptor tyrosine kinase, is highly active in malignant melanoma and displays a constitutive autophosphorylation activity possibly due to an activating mutation in its extracellular or transmembrane domain. In the focus formation assay the HER-Xmrk chimera is a potent transforming protein equivalent to other tyrosine kinase oncoproteins

Alternative splicing generates a novel isoform of the rat metabotropic GABA(B)R1 receptor

Here we present a novel isoform of the metabotropic G-protein-coupled receptor for gamma-aminobutyric acid (GABA). The isoform, termed GABA(B)R1c (R1c), differs from the recently identified R1a and R1b receptors by an in-frame insertion of 31 amino acids between the second extracellular loop and the fifth transmembrane region. Analysis of the rat GABA(B)R1 gene demonstrates that the insertion is the result of an alternative splicing event within a 567-bp intron between exons 16 and 17. In situ hybridization in the rat brain shows a wide distribution of R1c transcripts and an overlap with the R1a and R1b transcripts. The highest mRNA levels are found in cerebellar Purkinje cells, cerebral cortex, thalamus and hippocampal CA1 and CA3 regions. Western blots and immunodetection of recombinant epitope-tagged receptors as well as [125I]CGP71872 photoaffinity labelling of cell membranes demonstrate that R1c is correctly expressed, although at a lower level than the previously identified isoforms. When coexpressed with the newly characterized GABA(B)R2, R1c functionally couples to G-protein-activated Kir3.1/3.2 channels in Xenopus oocytes and to PLC-activating chimeric G(alpha)qo subunits in HEK-293 cells with a similar EC50 for agonists. These data suggest that the R1c isoform represents a functional GABA(B)R in the rat brain

Presynaptic and postsynaptic localization of GABA(B) receptors in neurons of the rat retina

The recently cloned GABA(B) receptors were localized in rat retina using specific antisera. Immunolabelling was detected in the inner and outer plexiform layers (IPL, OPL), and in a number of cells in the inner nuclear layer and the ganglion cell layer. Double-labelling experiments for GABA (gamma-aminobutyric acid) and GABA(B) receptors, respectively, demonstrated a co-localization in horizontal cells and amacrine cells. Electron microscopy showed that GABA(B) receptors of the OPL were localized presynaptically in horizontal cell processes invaginating into photoreceptor terminals. In the IPL, GABA(B) receptors were present presynaptically in amacrine cells, as well as postsynaptically in amacrine and ganglion cells. The postnatal development of GABA(B) receptors was also studied, and immunoreactivity was observed well before morphological and synaptic differentiation of retinal neurons. The present results suggest a presynaptic (autoreceptor) as well as postsynaptic role for GABA(B) receptors. In addition, the extrasynaptic localization of GABA(B) receptors could indicate a paracrine function of GABA in the retina

Developmental changes of agonist affinity at GABABR1 receptor variants in rat brain

Recently, two N-terminal splice variants of the metabotropic receptor for GABA (gamma-amino-butyric acid) were cloned. Here, we describe an antiserum that recognizes the two receptor variants. We demonstrate that these proteins are identical with GABAB receptors that are photoaffinity labeled with [125I]CGP71872 in rat brain. The C-terminal epitopes recognized by the antiserum are conserved in several vertebrate species but not in chicken. No hints for the existence of additional closely related receptor subtypes or variants are found in double-labeling experiments with antibody and photoaffinity ligand. Western blot analysis reveals widespread expression of the GABABR1 receptor proteins in rat brain with the highest level of expression at early postnatal stages. The binding affinity of the GABAB receptor agonist L-baclofen at native R1a and R1b variants is similar. In early postnatal development the affinity at R1a and R1b is 10-fold lower than in adult brain and gradually increases with aging

Ontogenic expression of anterior pituitary GABA(B) receptor subunits

gamma-Aminobutyric acid (GABA) is involved in the neuroendocrine control of hypophyseal secretion, acting both in the central nervous system and directly at the pituitary. We have characterized the properties of anterior pituitary GABA(B) receptors. In this work the ontogeny of rat anterior pituitary GABA(B) receptors and the pattern of subunit expression in rats of both sexes were determined. Western blot analysis showed a temporal decrease in GABA(B) subunits GABA(B(1a)) and GABA(B(1b)) expression in female anterior pituitary membranes from day 4 to adulthood, with GABA(B(1a)) being significantly more abundant than GABA(B(1b)) at early stages of development; the GABA(B(2)) subunit was barely detectable. In the male, GABA(B(1a)) followed a similar pattern and appeared to be significantly less abundant than in 4- and 12-day-old females; GABA(B(1b)) and GABA(B(2)) expression in the male was barely detectable. Scatchard plot analysis showed a temporal decrease in binding sites in female anterior pituitary membranes, in agreement with the western blot results. The number of binding sites was significantly higher in female than in male 4-day-old membranes. Dissociation constant values were similar for both sexes at all ages studied. This study reports for the first time the ontogeny of anterior pituitary GABA(B) receptors, showing a particular developmental pattern of subunit expression and a clear sexual dimorphism

Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding

The gamma-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain