IGF-I fusion proteins have distinct intracellular localization
The IGF-I chimeras used GFP as a fluorescent reporter (Fig. 2). The two signal peptides, MGKIS (exon 1 and 5' part of exon 3) and MITP (exon 2 and 5' part of exon 3), were fused to the N-terminal of GFP, while the B, C, A and variant E domains of IGF-I were fused to the C-terminal of GFP. This resulted in the expression of four different fusions: 1-G-3-4-6; 2-G-3-4-6; 1-G-3-4-5; and 2-G-3-4-5. When the constructions were expressed in cultured Hela cells, both signal peptides committed the GFP fluorescence to a cytoplasmic structures, consistent with the secretory apparatus (Fig. 3). However, a mixed localization pattern was seen for fusions containing the Eb-encoding exon 5, with a reproducible fluorescence seen in the nucleus and strongly in the nucleolus (Fig. 3). Such localizations were seen in multiple cells, in many independent transfections, with Hela cells from several sources, after shorter times of over-expression, after calcium phosphate and electroporation methods, and there was no correlation between levels of expression and localization (data not shown). Co-localization studies were performed using an anti-nucleolus monoclonal antibody and confirmed the localization as being nucleolar (Fig. 4).
A nucleolar localization signal in the Eb domain
To test the role of IGF-I domains in the nuclear and nucleolar localization, chimeras were constructed that deleted part of exon 3 encoding the B and C domains (2-G-4-5 and 1-G-4-5). The clear nuclear and nucleolar localization remained when the 2-G-4-5 construction was overexpressed (Fig. 5A). However, in the case of the 1-G-4-5 construction, the fluorescence was rapidly exported, and little nuclear or nucleolar localization was seen. We interpret this as an effect of the strength of the secretory signal peptide in competition with the nuclear and nucleolar localization signal(s). To exclude the possibility that the exon 2-encoded signal peptide was directly involved in the nuclear and nucleolar localization, it was deleted from the construction to make G-4-5, which still had a nuclear and nucleolar localization (Fig. 5A). To test the part of the Eb domain with this property, a naturally occurring splice variant, Ec, was used. This splice consists of the 5' portion of exon 5, encoding the N-terminal part of the Eb domain, and then splices to exon 6 [7]. This fusion (G-4-5-6) located throughout the cell, in a diffuse pattern, both in the nucleus and cytoplasm, without the striking nucleolar localization (Figure 5A). These data are consistent with the presence of a nucleolar localization signal at the C-terminal of the Eb domain.
The E peptides are not cleaved intracellularly
We performed western blotting on lysates from transfected cells (Fig. 5B). The size of the band for G-4-5 (lane 2) was larger than G-4-5-6 (lane 3), while G-4-6 (lane 4) was the smallest. This is compatible with the different sizes of the E domains and shows that the E domains are not cleaved intracellularly. This is despite the presence of previously mapped [8] prohormone processing sites and flanking residues in the chimeric proteins. However, the signal peptides appear to be cleaved, as 1-G-4-5 and 2-G-4-5 gave a band of the same size as G-4-5 (compare lanes 5 and 6 to lane 2). Note the band for 1-G-4-5 was faint on this exposure due to some degradation, but was better seen on overexposed blots (not shown).