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Localization of myopodin isoforms in eukaryotic cells
Myo1–Myo3 were stably expressed as V5 epitope tagged proteins in various cell lines by lentiviral transduction to investigate their expression and localization. To compare our data with previous studies we also analyzed the expression/localization of a variant of Myo1, Myo1Δ395 or MFKK-VVEE (Fig. 4A), that starts at M396, located 2 Amyloid Beta-peptide (25-35) before the nuclear localization sequence (NLS1, [4]) (MFKKRRRRARK). This construct is considered by others as the bona fide human myopodin protein that is expressed in eukaryotic cells [6]. To investigate a role for the PDZ domain in targeting myopodin to subcellular regions, we included a second Myo1 variant, termed Myo1Δ169 or MPDS-VVEE (Fig. 4A), that begins at M170 and lacks the N-terminal PDZ domain.
Fig. 4.
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Discussion
In this study, we demonstrate for the first time that at least three different myopodin transcripts are present in several eukaryotic cells. The Myo2 isoform was not predicted as an authentic transcript in public database repositories. The three transcripts give rise to three myopodin proteins that share an identical N-terminal region (harboring a PDZ domain), but are dissimilar at their C-terminus. Stable overexpression of these isoforms in different cell lines confirmed that they are translated and expressed as full length proteins.