The Amino-Terminus of Nitric Oxide Sensitive Guanylyl Cyclase α1 Does Not Affect Dimerization but Influences Subcellular Localization

Jan R Kraehling,Soenke Behrends,Nadine Haase,Mareike Busker,Tobias Haase,Markus Koglin,Monika Linnenbaum,Andreas Hofmann

doi:10.1371/journal.pone.0025772

Abstract

BackgroundNitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme formed by an α- and a β1-subunit. A splice variant (C-α1) of the α1-subunit, lacking at least the first 236 amino acids has been described by Sharina et al. 2008 and has been shown to be expressed in differentiating human embryonic cells. Wagner et al. 2005 have shown that the amino acids 61–128 of the α1-subunit are mandatory for quantitative heterodimerization implying that the C-α1-splice variant should lose its capacity to dimerize quantitatively.Methodology/Principal FindingsIn the current study we demonstrate preserved quantitative dimerization of the C-α1-splice by co-purification with the β1-subunit. In addition we used fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) using fusion proteins of the β1-subunit and the α1-subunit or the C-α1 variant with ECFP or EYFP. Analysis of the respective combinations in HEK-293 cells showed that the fluorescence lifetime was significantly shorter (≈0.3 ns) for α1/β1 and C-α1/β1 than the negative control. In addition we show that lack of the amino-terminus in the α1 splice variant directs it to a more oxidized subcellular compartment.Conclusions/SignificanceWe conclude that the amino-terminus of the α1-subunit is dispensable for dimerization in-vivo and ex-vivo, but influences the subcellular trafficking.

Highlights

Nitric oxide sensitive guanylyl cyclase is the physiological receptor for nitric oxide (NO) and nitric oxide releasing drugs
It has recently been shown that the a1-subunit of the nitric oxide receptor Nitric oxide sensitive guanylyl cyclase (NOsGC) undergoes splicing regulation in differentiating human embryonic cells [5]
The results indicated high levels of an amino-terminally deleted C-a1-splice form in differentiating cells that showed a different intracellular distribution in comparison to the canonical full-length a1-subunit

Summary

Introduction

Nitric oxide sensitive guanylyl cyclase is the physiological receptor for nitric oxide (NO) and nitric oxide releasing drugs. Since the occurrence of this splice variant has been linked to differentiation of human embryonic stem cells [5], the capacity of the C-a1 splice variant to heterodimerize and form a functionally active enzyme, is of biological importance. With the second experimental approach we examined fluorescence resonance energy transfer (FRET) based on fluorescence lifetime imaging (FLIM) in intact cells using fusion proteins of the respective subunits with fluorescent proteins. We observed that the fluorescent fusion of the C-a1 splice subunit is directed to a more oxidized subcellular compartment, while the respective C-a1/b1 heterodimer shows a cytosolic and nuclear localization

Results

Discussion

Findings

Materials and Methods