Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

Abstract

Beta-dystroglycan (beta-DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of beta-DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of beta-DG, characterizing a functional nuclear localization signal (NLS) in the beta-DG cytoplasmic domain, within amino acids 776-782. The NLS either alone or in the context of the whole beta-DG protein was able to target the heterologous GFP protein to the nucleus, with site-directed mutagenesis indicating that amino acids R(779) and K(780) are critical for NLS functionality. The nuclear transport molecules Importin (Imp)alpha and Impbeta bound with high affinity to the NLS of beta-DG and were found to be essential for NLS-dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of beta-DG may result in cytoplasmic retention, with Y(892) playing a key role. beta-DG thus follows a conventional Impalpha/beta-dependent nuclear import pathway, with important implications for its potential function in the nucleus.

Nuclear targeting of dystroglycan promotes the expression of androgen regulated transcription factors in prostate cancer

“…In order to test more rigorously a role for dystroglycan in the nucleus we used a construct comprising an n-terminal myristoyl-tag, dystroglycan cytoplasmic domain and c-terminal GFP tag (Myr-cβDG-GFP23). We hypothesised that such a construct should effectively mimic the properties of 31 kDa fragment of dystroglycan in that it is membrane associated, contains the complete cytoplasmic domain sequence including NLS24 and it has a GFP tag to allow efficient visualisation and cell sorting (Figure 5ABC). Furthermore this construct was efficiently translocated to the nucleus without additional stimulation with androgens thus allowing any transcriptional response to dystroglycan alone to be detected without the high background of an androgen-mediated transcriptional response.…”

“…Furthermore this construct was efficiently translocated to the nucleus without additional stimulation with androgens thus allowing any transcriptional response to dystroglycan alone to be detected without the high background of an androgen-mediated transcriptional response. In order to compare the effects of nuclear targeting on transcription, we designed a construct (Myr-cβDGΔNLS-GFP) mutated in the NLS1424 to prevent translocation to the nucleus. Immunofluorescence analysis of LNCaP cells transiently transfected with these constructs and enriched by FACS (Supplementary Figure 1) demonstrated that whilst Myr-cβDG-GFP had both plasma membrane and nuclear localisation, Myr-cβDGΔNLS-GFP had only membrane (and Golgi) localisation, with no nuclear localisation (Figure 5D).…”

“…Although the presence of an NLS on β-DG has been well documented2433 and the mechanisms underlying its entry into the nucleus are credible24, there is no comprehensive understanding of a function for the presence of dystroglycan in the nucleus thus far. It has been speculated that β-DG together with the DGC may offer stability to the nuclear membrane1634.…”

Dystrophin Dp71: The Smallest but Multifunctional Product of the Duchenne Muscular Dystrophy Gene

“…Recently, a functional NLS in the cytoplasmic tail of β-dystroglycan was identified [63], which makes it plausible to propose that Dp71 gains access to the nucleus through its association with β-dystroglycan. Finally, it is noteworthy that a Dp71f-like protein was observed in the mitochondria of mdx and mdx 3cv mice [64].…”

Dystrophin–Glycoprotein Complex in Blood Cells

“…Due to the presence of a conventional nuclear localization sequence (NLS)-/Imp-dependent nuclear import pathway in the cytoplasmic juxtamembrane region of β-Dg [87], β-Dg and proteolytic fragments containing the nuclear localization signal can be targeted to the nucleus via an importin-dependent pathway [88], where it can exert efects on nuclear architecture [89].…”