GABA(B) receptor heterodimer-component localisation in human brain.

In recombinant cell lines, functional GABA(B) receptors are only formed by the heterodimerisation between two related G-protein coupled receptor proteins GABA(B)R1 (GBR1) and GABA(B)R2 (GBR2), whilst the individual GBR1 or GBR2 do not produce fully functional receptors. To determine whether the heterodimerisation occurs in vivo, novel polyclonal antibodies targeting the C termini of GBR1 and GBR2, were raised in different species, characterised, and used to determine the relative localisation of the reported heterodimer components in human brain tissue, using immunohistochemistry. The use of different species for the raising of the antisera allowed double immunofluorescent labelling of the receptors as an indication of GBR1/GBR2 receptor co-localisation in human brain. The presence of both proteins is reported in cerebellum, hippocampus, cortex, thalamus and basal ganglia. Regions of the brainstem including pons and medulla, also express GBR1 and GBR2 protein. The double immunofluorescence demonstrated that GBR1 and GBR2 are co-localised in the human cerebellar cortex. Together these results suggest the widespread distribution of GABA(B) receptors in human brain, and that GABA(B) receptors GBR1 and GBR2 can exist in the same cell, and therefore may function as a heterodimer in the human brain.

Heterodimerization is required for the formation of a functional GABA(B) receptor.

GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABA(A/C)) receptors to produce fast synaptic inhibition and metabotropic (GABA(B)) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABA(B) receptor (GABA(B)R1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABA(B)R2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABA(B)R2-receptor subtype forms heterodimers with GABA(B)R1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABA(B)R2 in HEK293T cells, GABA(B)R1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABA(B) receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABA(B) receptors may be heterodimers composed of GABA(B)R1 and GABA(B)R2.

The product of varicella-zoster virus gene 62 autoregulates its own promoter.

Varicella-zoster (VZV) gene 62 encodes a protein with a predicted Mr of 140,000 (140K) which has considerable amino acid identity with the major immediate early (IE) protein Vmw175 (ICP4) of herpes simplex virus type I (HSV-1). Vmw175 is an essential virus polypeptide with a pivotal role in the activation of early and late viral gene expression and also in the repression of IE gene expression. The VZV 140K protein has been shown to function as a strong transcriptional activator in transfection assays and largely complements for the loss of Vmw175 function in HSV-1. We report the results of cotransfection experiments which demonstrate that the 140K protein strongly represses expression from its own promoter, that of gene 62, thus establishing further functional similarity between it and Vmw175. However, whereas Vmw175 can substitute for the 140K protein in repression of the gene 62 promoter, the 140K protein does not repress the HSV-1 IE3 promoter in the reciprocal experiment. The integrity of a domain of Vmw175 (designated region 2), previously shown to be crucial for repression of the HSV-1 IE3 promoter, is also required for repression of the gene 62 promoter. Moreover, a similar requirement for the highly similar region 2 of the 140K protein for repression is demonstrated, suggesting that VZV 140K protein and HSV-1 Vmw175 autoregulate IE gene expression by a related mechanism.

A herpes simplex virus type 1 recombinant with both copies of the Vmw175 coding sequences replaced by the homologous varicella-zoster virus open reading frame.

Varicella-zoster virus (VZV) gene 62 encodes a protein with a predicted Mr of 140,000 (VZV 140K) that shares considerable amino acid homology with the immediate early (IE) regulatory protein Vmw175 of herpes simplex virus type 1 (HSV-1) and is believed to be its functional equivalent. We have tested this hypothesis by insertion of VZV gene 62 (expressed from the HSV-1 IE3 promoter) into both IE3 gene loci in the short region repeats of the HSV-1 genome. The parent virus used for this manipulation was D30EBA, which is a variant of HSV-1 from which the majority of the Vmw175 coding sequences have been deleted. Like other HSV-1 viruses lacking Vmw175 functions, D30EBA is able to grow only in cell lines which express Vmw175 constitutively. The resulting recombinant virus. HSV-140, is able to propagate (but unable to form obvious plaques) on normal cell lines. The properties of HSV-140 were studied by monitoring the time course of polypeptide expression and DNA replication during normal infection. We found that at high multiplicity HSV-140 synthesized apparently normal amounts of many viral polypeptides but that the expression of certain late genes was reduced; this slight defect may be related to less efficient DNA replication by HSV-140. At low multiplicity HSV-140 expressed viral proteins inefficiently. Surprisingly, VZV 140K was produced in large amounts at later times of a normal infection, indicating that the polypeptide fails to autoregulate the IE3 promoter. The results strongly suggest that VZV 140K is able to perform most of the functions of Vmw175 during growth of HSV-1, but that differences in detail lead to less efficient virus growth.

Control of expression of the varicella-zoster virus major immediate early gene.

The cis-acting DNA sequences and trans-acting proteins that control the expression of the major immediate early (IE) gene of varicella-zoster virus (VZV) were investigated. The location of the IE mRNA 5' terminus was determined by primer extension and S1 nuclease analyses and the functional activities of DNA sequences upstream of this site were analysed by a transfection assay. The VZV IE promoter exhibited low activity in BHK and HeLa cells, but was transactivated by the herpes simplex virus type 1 (HSV-1) virion protein Vmw65. DNA sequences between positions -131 and +57 were responsible for promoter activity, whereas sequences between -410 and -131 mediated the response to Vmw65. Two short elements in the -410 to -131 region formed protein-DNA complexes with HeLa cell nuclear proteins and formed a ternary complex when Vmw65 was added. One of the elements, ATGTAAATGAAAT, possessed a strong similarity to the HSV-1 TAATGARAT. The VZV homologue of Vmw65, encoded by open reading frame (ORF) 10, failed to trans-activate expression from HSV-1 or VZV IE promoters and did not form a ternary complex with functional TAATGARAT elements and HeLa cell proteins. Therefore, stimulation of VZV IE transcription by Vmw65 can occur by a mechanism similar to that employed by HSV-1, but VZV ORF 10 does not function as a trans-activator of IE gene expression.