The role of neuregulin 1-β3 in the nucleus Free

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Introduction

The neuregulins (NRG) are members of the ErbB family of growth factors, which bind to and activate the ErbB3 (HER3) and/or ErbB4 (HER4) receptors. These growth factors are usually present on the cell surface or are secreted proteins, but recently NRG1-β3 tagged with enhanced green fluorescent protein has been visualized either in the nucleus in nucleoli or in SC35 positive spliceosomes (Golding et al., 2004) using low light digital microscopy and this localization was independent of the presence of its receptors. The first 21 amino acids of the protein have been shown to be responsible for localization to nucleoli and a 30 amino acid region (residues 49-79) of NRG1-β3 was responsible for localization to spliceosomes. In this work we have used site directed mutagenesis to identify specific residues in these two regions responsible for localization to these two distinct sub-nuclear structures.

Methods

We have performed site directed mutagenesis on residues 14-16 (KKK) of NRG1- β3 which represents a possible nucleolar localization sequence and constructed a number of deletion mutants within residues 49-79 which are responsible for localization to splicing speckles. These were ligated into GFP constructs and verified by sequencing. Constructs were transfected into Cos7 cells and visualized to determine their localization using low light digital fluorescence microscopy.

Results and Conclusions

Site directed mutagenesis of residues 14-16 revealed that a single mutation of lysine to alanine (N21AKK, N21KAK and N21KKA) did not localize to nucleoli. Substitution of lysine for alanine at the third position in the KKK sequence within the putative NLS, when in conjunction with a further substitution of either of the first two positions hindered nucleolar localisation. When a range of deletion mutants within residues 49-79 were expressed in Cos7 cells these all hindered the localization of NRG1- β3 in the nucleus to spliceosomes.

Further site directed mutagenesis of the 49-79 region is underway to determine the specific residues responsible for localization. We are also exploring the functional significance of nuclear localization of NRG1. Nucleoli and SC35 positive speckles contribute to ribosome synthesis, transcriptional control and RNA splicing respectively and we are testing if these are altered in the presence of the growth factor.