title: Quantification of G protein Gaalphas subunit splice variants in different human tissues and cells using pyrosequencing.
abstract: The G protein Galphas is derived from four alternatively spliced transcripts, two long variants (Galphas(L)+CAG and Galphas(L)-CAG), which include an extra 45-bp segment, and two short variants (Galphas(S)+CAG and Galphas(S)-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 Galphas transcript ratios in various human tissues and cells. Galphas(S)/Galphas 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 Galphas(S)+CAG ratios, which ranged from a total Galphas 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 Galphas(S)-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 Galphas(L)+CAG ratio ranged from 0.02 +/- 0.02 in heart tissue to 0.05 +/- 0.01 in retinoblastoma cells, with a varying proportion of Galphas(L)-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 Galphas in different tissues affected Galphas(L)-CAG and Gas(S)+CAG rather than Galphas(L)+CAG and Galphas(S-)CAG. Furthermore, stimulation of cells seemed to affect splice variant ratios. These results are, therefore, suggestive of different biological functions of these variants.
authors.affiliation: Department of Pharmacology, University Hospital Essen, D-45122 Essen, Germany. ulrich.frey@uni-essen.de
authors.forename: Ulrich H
authors.initials: UH
authors.lastname: Frey
authors.forename: Holger
authors.initials: H
authors.lastname: Nückel
authors.forename: Dobromir
authors.initials: D
authors.lastname: Dobrev
authors.forename: Iris
authors.initials: I
authors.lastname: Manthey
authors.forename: I E
authors.initials: IE
authors.lastname: Sandalcioglu
authors.forename: Andreas
authors.initials: A
authors.lastname: Eisenhardt
authors.forename: Karl
authors.initials: K
authors.lastname: Worm
authors.forename: Hans
authors.initials: H
authors.lastname: Hauner
authors.forename: Winfried
authors.initials: W
authors.lastname: Siffert
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D001921:Brain / Q000378:metabolism / Q000473:pathology; D001940:Breast / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D005260:Female; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008214:Lymphocytes / Q000187:drug effects; D008297:Male; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D012175:Retinoblastoma / Q000378:metabolism / Q000473:pathology; D017422:Sequence Analysis, DNA / Q000379:methods*; D014018:Tissue Distribution; D014158:Transcription, Genetic; D001749:Urinary Bladder Neoplasms / Q000378:metabolism / Q000473:pathology
chemical_list: D012333:RNA, Messenger; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol