- Research article
- Open Access
Nucleolar localization of an isoform of the IGF-I precursor
© Tan et al; licensee BioMed Central Ltd. 2002
- Received: 13 April 2002
- Accepted: 2 July 2002
- Published: 2 July 2002
Alternative exons encode different isoforms of the human insulin-like growth factor-I (IGF-I) precursor without altering mature IGF-I. We hypothesized that the various IGF-I precursors may traffic IGF-I differently. Chimeric IGF-I precursors were made with green fluorescent protein (GFP) cloned between the signal and mature IGF-I domains.
Chimeras containing exons 1 or 2 were located in the cytoplasm, consistent with a secretory pathway, and suggesting that both exons encoded functional signal peptides. Exon 5-containing chimeras localized to the nucleus and strongly to the nucleolus, while chimeras containing exon 6 or the upstream portion of exon 5 did not. Nuclear and nucleolar localization also occurred when the mature IGF-I domain was deleted from the chimeras, or when signal peptides were deleted.
We have identified a nucleolar localization for an isoform of the human IGF-I precursor. The findings are consistent with the presence of a nuclear and nucleolar localization signal situated in the C-terminal part of the exon 5-encoded domain with similarities to signals in several other growth factors.
- Green Fluorescent Protein
- Signal Peptide
- Chimeric Protein
- Nucleolar Localization
- Upstream Portion
IGF-I fusion proteins have distinct intracellular localization
A nucleolar localization signal in 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  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).
Using GFP-fusions, we found that the IGF-I exon 5-encoded Eb domain localized chimeric proteins to the nucleus and nucleolus. The localization did not require the signal peptides, mature peptide or the N-terminal part of the Eb peptide. The last four amino acid residues of the Eb domain matches the motif R/K-R/K-X-R/K which is found in several nucleolar localization signals . The presence of the chimeric IGF-I precursors in the nucleus and nucleolus was not an artefact of over-expression since it was seen in cells with varying expression levels and at shorter times after transfection. It is unlikely that this localization was an artefact of tagging because of the consistent pattern seen in different chimeric contexts. Nuclear and nucleolar localization were seen more in the context of the exon 2-encoded, rather than the exon 1-encoded signal peptide, but the signal peptide itself was not required for such localization. This may reflect an indirect action of the signal peptide. Thus, there may be a relative weakness of secretory function for the exon 2-encoded signal peptide compared to that encoded by exon 1. Our data suggest that the signal peptides were efficiently cleaved from the chimeras. The Eb domain was not cleaved from the chimeric precursor IGF-I, and we propose that any biological activity in the nucleolus may be a property of pro-IGF-I. The final location of the chimeras may represent a balance of the competition between secretory and nuclear/nucleolar localization signals.
The nucleolus is a membrane-less dynamic structure organized by transcription and disassembled during mitosis . There are a number of examples of nucleolar localization of growth factors . A well-characterized example is fibroblast growth factor-3, where two nuclear and one nucleolar localization signals act in concert  and in competition with secretory signals , with an inhibitory action on cell growth . Recently a nucleolar binding protein for fibroblast growth factor-3, NoBP, has been isolated and characterized . A question is whether other nucleolar growth factors also interact with NoBP, or whether they interact through distinct proteins. The nucleolus has multiple functions, including ribosome synthesis, signal recognition particle processing and assembly, and processing of U6 snRNP, snoRNAs, telomerase RNA and tRNAs . The nucleolus is a key site in the regulation of the oncogenic p53-Mdm2-ARF pathway . It is not clear how an isoform of the IGF-I may affect any of these nucleolar functions.
We found that the isoform of the human IGF-I precursor encoded by exon 5 localized to the nucleus and strongly to the nucleolus. Precursors containing exon 6 or the upstream portion of exon 5 did not. Nuclear and nucleolar localization also occurred when the mature IGF-I domain was deleted from the chimeras, or when signal peptides were deleted. The findings are consistent with the presence of a nuclear and nucleolar localization signal situated in the C-terminal part of the exon 5-encoded domain. These findings are similar to the localization of several other growth factors in the nucleolus.
Construction of chimeric proteins
The chimeric constructions are shown in Fig. 2. Coordinates are given relative to the N-terminal residue (G) of the mature IGF-I peptide. IGF-I exon 1 encodes part of a signal peptide from residues MGK [-48 to -46] to K [-28]. Exon 3 encodes the remainder of the signal peptide from residues VKM [-27 to -25] to A [-1]. This was cloned upstream of the cDNA for GFP. Exon 3 encodes the mature IGF-I peptide starting at residues GPET [1–4] to F , exon 4 encodes the mature IGF-I peptide from residue N  to A  and part of the E domain from residues RSV [71–73] to K . Exon 6 encodes an alternative Ea domain starting at residues FVH [87–89] to M . This IGF-I cDNA sequence is from Genbank X57025 . In other chimeras, exon 2 replaces exon 1 and encodes the residues MITPT [-32 to -28]. The sequence is from Genbank M37484 . Exon 5 encodes an alternative Eb domain starting at residues YDP [87–89] to K . This sequence is from Genbank M11568 . Exon 5–6 encodes the alternative Ec domain, beginning with exon 5-encoded residues YDP [87–89] to K , then ending in the exon 6-encoded residues GMTFEERK [103 to 110]. This sequence is from Genbank U40870 .
Signal peptides were made by PCR using primers AgeI-IGF-MGKISS (5'-GGCTACCGGTCTTCAGAAGCAATGGGAA) or AgeI-IGF-MITP (5'-GGCTACCGGTATGATTACACCTACAGTGAA) with AgeI-IGF-SPA (5'-GGCTACCGGTCCAGCCGTGGCAGA) and cloned into the Age I site of pEGFP-C1. This cloning strategy introduces four amino acid residues (PVAT) between the last residue A [-1] of the signal peptide and the first residue of GFP. The mature IGF-I and Ea, or Eb or Ec domains were generated by PCR using primers Eco-GPETLC (5'-GGAATTCCGGACCGGAGACGCTCTGC) and Sac-E6-650A (5'-GCTGAGCCGCGGTTCACTCCTCAGGAGGGTCTT) or Sac-E5-543A (5'-GCTGAGCCGCGGTTTAATCCTCCTGTCCTTCA) or SacII-E6-619A (5'-GCTGAGCCGCGGGTAGTTCTTGTTTCCTGC). The PCR products were cloned into the Eco RI and Sac II sites in pEGFP-C1. The chimeras with IGF-I exon 3 deletions were made using the primer EcoRI-MOD4-2244S (5'-GGAATTCCGGGTATGGCTCCAGCAGT) resulting in fusions between GFP and IGF-I from residues GYG [30–32]. Eco RI and Sac II cloning sites in pEGFP-C1 were used. All PCR products were made by proof-reading PCR using Pfu Turbo (Stratagene) and all clones were verified by sequencing.
Transfections, cell imaging and western blotting
2 ug of each plasmid was transfected into Hela cells cultured on coverslips in 3 cm wells using 94 ul of Fugene 6 (Boehringer). After 12–24 hours of expression, cells were incubated at room temperature for one hour and fixed in 2% w/v paraformaldehyde. Cells subjected to immunofluorescence were washed in PBS, permeabilized with 0.2% v/v Triton X100 in 1% v/v normal goat serum, before incubation with an anti-human nucleolus monoclonal antibody (Calbiochem). After washes in 1% normal goat serum, cells were incubated with a Texas-red-labeled goat anti-mouse antibody (Jackson Labs). All coverslips were mounted on glass slides and imaged with a fluorescent microscope (Leica DMR) and a charge-coupled device camera (Leica DC200). For western blotting, cells from 3 cm wells were harvested in Laemmli buffer containing 6 M urea. 10 ug of total cellular protein was loaded onto a 12% SDS-PAGE, then semi-dry blotted onto Hybond C+ (Amersham) before blocking and incubation with monoclonal anti-GFP antibodies (Boehringer).
We are grateful to C. Camacho-Hubner for helpful discussions and M. D. Turner for comments on the manuscript. The work was partly supported by the Wellcome Trust (045401).
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