Gorrie, George Henry; (1997) The development and application of the Semliki Forest virus expression system to the study of GABAA receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

GABAA receptors are presumed to be pentameric hetero oloigomers composed of subunits potentially drawn from five subunit classes with multiple members; α(1-6), β (1-4), [gamma] (1-3), δ, and ԑ. The mechanisms by which neurones assemble this potentially large number of subunits into functional GABAA receptors is unknown. Heterologous expression systems have demonstrated that particular combinations of GABAA subunits assemble to form channels whilst others are selectively retained within the endoplasmic reticulum of the cell. In order to determine whether the assembly rules which have been defined in such heterologous expression system are representative of the assembly of GABAA receptor subunits within primary cultures of neurones, a viral expression system was used to express recombinant epitope tagged GABAA subunits in neurones. The viral expression system which was chosen was based upon the Semliki Forest virus. This particular viral expression system was chosen because of its ability to infect neurones as well as many other cell types with high efficiency. Recombinant viral particles were produced which could direct the synthesis of the α1 (9E10), β2 (9E10), [gamma]2 L FLAG and [gamma]2 S (9E10) tagged subunits. Using the Semliki viral expression system the assembly of αl (9E10) and β2 (9E10) subunits in BHK cells was compared to that produced when these subunits were transiently transfected into HEK 293 cells. The results of the viral infections replicated the assembly work performed using conventional transient transfections of HEK 293 cells. Expression of individual αl (9E10) or β2 (9E10) subunits resulted in their selective retention within the endoplasmic reticulum of the cells whilst on co-expression both the αl (9E10) and β2 (9E10) subunits could access the cell surface. Whole cell electron microscopy of such cells demonstrated that the subunits were on the surface of the cell and were preferentially associated with the microvilli of the BHK cells. The powerful nature of the Semliki viral expression system allowed the demonstration of a biophysical association between α1 (9E10) and β2 (9E10) proteins as demonstrated by changes in the sedimentation coefficients of the two proteins on co-infection of BHK cells by the two viral species. When individually expressed the α1 (9E10) and β2 (9E10) proteins have 5S sedimentation coefficients. On co-infection the sedimentation coefficients of both proteins shifted to 9S. For the first time, the viral expression system enabled the efficiency of the assembly of the α1 (9E10) and β2 (9E10) subunits to be estimated. It also provided a means to study the stability of assembled and unassembled receptor composed of α1 (9E10) and β2 (9E10) subunits. It was found that only 33% of the available α1 (9E10) and β2 (9E10) subunits became incorporated into the assembled 9S complex. Once formed, this 9S sedimenting species was relatively stable only dropping 10[percent] of its initial signal over a 16 hour period. In contrast the unassembled receptor was quickly degraded, with a 75[percent] drop over 6 hours. The half life of individually virally expressed α1 (9E10) and β2 (9E10) subunits was also investigated and determined to be between 2 to 3 hours. The stoichiometry of a GABAA receptor composed of α1 (9E10) and β2 (9E10) receptors was also evaluated using the Semliki expression system. Metabolic labelling in combination with 9E10 immunoprecipitaiton of the assembled 9S receptor allowed a determination of the ratio of α to β subunits after correction for the relative methionine contents of the two proteins. The ratio of α1 (9E10) to β2 (9E10) subunits was 1:1. The toxicity of viral infection upon SCG neurones was studied by infecting SCG neurones with a recombinant SFV virus engineered to make the Green Fluorescent Protein and then subsequently recording the resting membrane potential of neurones which could produce green fluorescence. The neurones which produced green fluorescence had normal resting potentials and a normal ability to produce action potentials. Knowing that the viral expression system had limited toxicity to the SCG neurones and that the expression system was capable of supporting the assembly of the α1 (9E10) and β2 (9E10) subunits within BHK cells the assembly of these subunits was studied in primary cultures of SCG neurones. Infection of such cultures by either the α1 (9E10) or β2 (9E10) virus particles resulted in the retention of the GABAA subunit within the cell body of the neurone. On co-infection marked surface expression could be demonstrated. This indicated that a common assembly mechanism occurs between neurones and transformed cell lines, with retention within the endoplasmic reticulum and subsequent degradation featuring as a major control mechanism. The biochemistry, and cell biology of the [gamma]2 subunit was also investigated using the Semliki expression system. The [gamma]2 S (9E10) protein was demonstrated to behave differently to the long isoform on polyacrylamide gel electrophoresis (PAGE) and sucrose gradient sedimentation studies. The size of the long isoform on PAGE was 45KDa which contrasted with the 50KDa size for the [gamma]2 S (9E10). The sucrose gradient studies indicated that the [gamma]2 S (9E10) protein had a sharp profile peaking at 3S whilst the [gamma]2 L FLAG had a much broader profile with its peak nearer 4S and aggregates between 5 and 7S. Viral expression of the short and long splice variants of the [gamma]2 subunit within BHK cells and SCG neurones produced contrasting cell biology. The short variant was shown to be capable of trafficking to the surface of BHK cells and into the processes of SCG neurones. The short splice variant was also capable of being endocytosed from the surface of BHK cells. Electron microscopic examination of BHK cells infected with the [gamma]2 S (9E10) virus was used to demonstrate the association of the [gamma]2 S (9E10) protein with coated pits. In contrast the long variant was not able to access the plasma membrane when individually expressed. Expression of the long isoform of the [gamma]2 subunit within SCG neurones produced only a cell body pattern of expression consistent with the retention of the subunit within the endoplasmic reticulum. Finally the capability of the [gamma]2 subunit to be tyrosine phosphorylated within cells expressing high levels of the tyrosine kinase Src was tested. When transiently coexpressed with α 1, β 1 and v-Src both the [gamma]2 L (9E10) and [gamma]2 S (9E10) proteins could be shown to undergo phosphorylation specifically at tyrosine residue 365 and 367 in the short and 373 and 375 in the long.

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