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Quantification of G Protein Gαs Subunit Splice Variants in Different Human Tissues and Cells Using Pyrosequencing

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The G protein Gαs is derived from four alternatively spliced transcripts, two long variants (GαsL+CAG and GαsL−CAG), which include an extra 45-bp segment, and two short variants (GαsS+CAG and GαsS−CAG). The long and short forms differ in each case by splicing in or out of a serine residue encoded at the 3′ end of the variable exon 3. The relative expression of all four variants in human tissues is poorly investigated due to experimental limitations. We therefore established a method for reliable relative mRNA quantification of these splice variants based on the Pyrosequencing technology, and determined Gαs transcript ratios in various human tissues and cells. GαsS/Gαs ratio was highest in blood mononuclear cells (0.84 ± 0.02, n = 16) and lowest in the brain (0.51 ± 0.14, n = 3). The different ranges resulted from differences in GαsS+CAG ratios, which ranged from a total Gαs ratio of 0.32 ± 0.07 (n = 12) in heart tissue to 0.57 ± 0.03 (n = 16) in blood mononuclear cells (p < 0.0001), whereas the GαsS−CAG ratio was rather constant and ranged from 0.22 ± 0.04 (n = 7) in retinoblastoma cells to 0.27 ± 0.04 in lymphocytes (p = 0.19). The GαsL+CAG ratio ranged from 0.02 ± 0.02 in heart tissue to 0.05 ± 0.01 in retinoblastoma cells, with a varying proportion of GαsL−CAG, which ranged from 0.14 ± 0.02 in blood mononuclear cells to 0.41 ± 0.08 in heart tissue. Stimulation of immortalized B lymphoblasts with isoproterenol resulted in significant changes of splice variant ratios. Our data indicate that changes of long and short ratios of Gαs in different tissues affected GαsL−CAG and GαsS+CAG rather than GαsL+CAG and GαsS−CAG. Furthermore, stimulation of cells seemed to affect splice variant ratios. These results are, therefore, suggestive of different biological functions of these variants.
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Keywords: G proteins; Pyrosequencing; Quantification; Signal transduction; Splice variants

Document Type: Review Article

Affiliations: 1: Institut für Pharmakologie, Universitätsklinikum, D-45122 Essen, Germany 2: Institut für Pharmakologie, Universitätsklinikum, D-45122 Essen, Germany, Klinik für Hämatologie, Universitätsklinikum, D-45122 Essen, Germany 3: Institut für Pharmakologie und Toxikologie, D-01307 Dresden, Germany 4: Klinik für Neurochirurgie, Universitätsklinikum, D-45122 Essen, Germany 5: Klinik für Urologie, Universitätsklinikum, D-45122 Essen, Germany 6: Institut für Pathologie, Universitätsklinikum, D-45122 Essen, Germany 7: Else-Kröner-Fresenius-Zentrum für Ernährungsmedizin, Technische Universität München, D-81675 München, Germany

Publication date: February 1, 2005

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