Identifying the G protein, Gz alpha, and its associated proteins in nervous tissue using mass spectrometry and microsequencing techniques.
The signaling pathway associated with pertussis and cholera toxin sensitive G proteins have been extensively investigated. In contrast, the function and associated signal transduction cascade for the pertussis toxin insensitive G protein, Gz alpha have remained elusive. Therefore, the aim of this study was to identify the signal transduction pathway associated with Gz alpha by using the protein identification techniques of matrix assisted laser desorption ionization-time of flight mass spectroscopy and N-terminal Edman sequencing. We have chosen this technique to identify proteins that Gz alpha associates with and to gain insights into the potential role this G protein plays in cells. As Gz alpha is predominantly localized in neuronal tissues, homogenates of whole brain tissue were used. Gz alpha and its associated proteins were immunoprecipitated from brain tissue and identified. The immunoprecipitation of four proteins (140, 46, 41 and 36 kDa) was shown to be inhibited in the presence of the Gz alpha peptide. These proteins were subsequently identified as phospholipase C (PLC)-gamma, beta or gamma-actin, Gz alpha and G beta, the beta subunit of heterotrimeric G proteins, respectively. These results suggest that Gz alpha exists in a protein complex with the actin cytoskeleton and an important intracellular signalling enzyme, PLC-gamma. These methods are powerful techniques for determining protein-protein interactions, and provide the first step to the identification of signalling proteins that Gz alpha associates with. However further experimentation will be required to determine the biological relevance of these protein interactions.
Bartlett
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SE
Division of Neuroscience, The John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia.
Hendry
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D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001923:Brain Chemistry; D002851:Chromatography, High Pressure Liquid; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000737:chemistry* / Q000276:immunology; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000276:immunology; D010446:Peptide Fragments / Q000032:analysis; D011233:Precipitin Tests
D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D019204:GTP-Binding Proteins