Premium
Cloning and characterisation of GIRK1 variants resulting from alternative RNA editing of the KCNJ3 gene transcript in a human breast cancer cell line
Author(s) -
Wagner Valerie,
Stadelmeyer Elke,
Riederer Monika,
Regitnig Peter,
Gorischek Astrid,
DeVaney Trevor,
Schmidt Kurt,
Tritthart Helmut A.,
Hirschberg Koret,
Bauernhofer Thomas,
Schreibmayer Wolfgang
Publication year - 2010
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.22564
Subject(s) - biology , g protein coupled inwardly rectifying potassium channel , xenopus , messenger rna , alternative splicing , microbiology and biotechnology , rna splicing , gene expression , gene , rna , signal transduction , genetics , g protein
The aim of this study was to investigate the impact of increased mRNA levels encoding GIRK1 in breast tumours on GIRK protein expression. mRNA levels encoding hGIRK1 and hGIRK4 in the MCF7, MCF10A and MDA‐MB‐453 breast cancer cell lines were assessed and the corresponding proteins detected using Western blots. cDNAs encoding for four hGIRK1 splice variants (hGIRK1a, 1c, 1d and 1e) were cloned from the MCF7 cell line. Subcellular localisation of fluorescence labelled hGIRK1a–e and hGIRK4 and of endogenous GIRK1 and GIRK4 subunits was monitored in the MCF7 cell line. All hGIRK1 splice variants and hGIRK4 were predominantly located within the endoplasmic reticulum. Heterologous expression in Xenopus laevis oocytes and two electrode voltage clamp experiments together with confocal microscopy were performed. Only the hGIRK1a subunit was able to form functional GIRK channels in connection with hGIRK4. The other splice variants are expressed, but exert a dominant negative effect on heterooligomeric channel function. Hence, alternative splicing of the KCNJ3 gene transcript in the MCF7 cell line leads to a family of mRNA's, encoding truncated versions of the hGIRK1 protein. The very high abundance of mRNA's encoding GIRK1 together with the presence of GIRK1 protein suggests a pathophysiological role in breast cancer. J. Cell. Biochem. 110: 598–608, 2010. © 2010 Wiley‐Liss, Inc.