Abstract
RGSZ1 has been reported to interact with G-protein subunits of the Galphai family and function as a GTPase-accelerating protein on intrinsic Galphai GTPase activity. This article describes several experimental approaches and assays used to investigate the effect of RGSZ1 on Galphai subunits. The formats described here include physical and functional interaction assays by which the association of RGSZ1 with Galphai is explored both in vitro and in vivo. The methods analyzing physical interaction include pull-down and coimmunoprecipitation assays. We also apply yeast two-hybrid techniques to detect RGSZ1 protein interaction with Galpha subunits. Additionally, we developed several functional assay systems to identify the functional relationship between RGSZ1 and Galphai, such as the single turnover GTPase assay, yeast pheromone response assay, mitogen-activated protein kinase assay, and serum response element reporter assay.
MeSH terms
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Animals
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CHO Cells
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Cell-Free System
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Cricetinae
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Dopamine Agonists / metabolism
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GTP Phosphohydrolases / metabolism
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GTP-Binding Protein alpha Subunits, Gi-Go / genetics
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GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
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GTPase-Activating Proteins / genetics
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GTPase-Activating Proteins / metabolism*
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Genes, Reporter
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Humans
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Membrane Proteins / genetics
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Membrane Proteins / metabolism*
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Mitogen-Activated Protein Kinases / metabolism
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Nerve Tissue Proteins / genetics
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Nerve Tissue Proteins / metabolism*
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PC12 Cells
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Pheromones / metabolism
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Quinpirole / metabolism
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RGS Proteins
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Rats
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Receptors, Dopamine D2 / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Serum Response Element
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Signal Transduction / physiology
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Sulfur Radioisotopes / metabolism
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Two-Hybrid System Techniques
Substances
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Dopamine Agonists
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GTPase-Activating Proteins
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Membrane Proteins
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Nerve Tissue Proteins
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Pheromones
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RGS Proteins
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RGS20 protein, human
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Receptors, Dopamine D2
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Recombinant Fusion Proteins
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Sulfur Radioisotopes
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Quinpirole
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Mitogen-Activated Protein Kinases
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GTP Phosphohydrolases
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GTP-Binding Protein alpha Subunits, Gi-Go