Nitric Oxide Activates the β2 Subunit of Soluble Guanylyl Cyclase in the Absence of a Second Subunit

Abstract

Previously characterized mammalian soluble guanylyl cyclases form ␣/␤ heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named ␣ 1 , ␤ 1 , ␣ 2 , and ␤ 2 . The ␣ 1 /␤ 1 and ␣ 2 /␤ 1 heterodimeric enzyme isoforms have been rigorously characterized. The role of the ␤ 2 subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the ␤ 2 subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the ␤ 3 subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mM free Mn 2؉ . The EC 50 values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the ␣ 1 /␤ 1 heterodimeric form. In the presence of the detergent Tween, NO sensitivity of ␤ 2 was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the ␤ 2 subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.

Structural and Functional Characterization of the Dimerization Region of Soluble Guanylyl Cyclase

“…It is tempting to speculate that expression of alternatively spliced variants or isoforms of the ␤ subunit exposing modified N-or C-terminal portions but retaining the dimerization region may contribute to the fine tuning of cellular cyclase activity through targeted degradation of the resultant complexes. Notably various isoforms of human and rat ␤ 2 expressed in kidney, liver, and brain (19,32) are characterized by N-terminal truncations of up to 79 residues and C-terminal extensions of up to 63 residues compared with the corresponding ␤ 1 subunits (19,33,34). Among these, the rat ␤ 2 isoform has been shown to reduce considerably the activity of coexpressed ␣ 1 ␤ 1 in vitro, and therefore enhanced expression of ␤ 2 has been implicated in the downregulation of renal guanylyl cyclase activity in Dahl salt-sensitive rats (35).…”

A Soluble Guanylate Cyclase Mediates Negative Signaling by Ammonium on Expression of Nitrate Reductase in Chlamydomonas

“…However, the expressed proteins already had a high GC activity so that additional NO activation could not be shown, possibly due to an inappropriate folding of this protein in the E. coli host that would release the GC domain from the control by NO. Additionally, another subunit might be required to make the GC domain further activated by NO as reported in other animal GCs (Koglin et al, 2001). Interestingly, CYG56 lacks three Cys residues conserved in other GCs (see Supplemental Figures 2 and 3 online).…”

Preliminary characterization of two atypical soluble guanylyl cyclases in the central and peripheral nervous system ofDrosophila melanogaster

“…Gyc-88E, Gyc89Db and CP12881 share with MsGC-β3 the absence of two specific cysteine residues in the N-terminal domain (equivalent to cys78 and cys214 in the rat β1 subunit), which have been found to be required for NO activation in α/β heterodimers involving the rat β1 subunit (Friebe et al, 1997). The rat β2 subunit, which is slightly NO sensitive (Koglin et al, 2001), does however retain these cysteine residues. Gyc-88E and CP12881 are similar to MsGC-β3 and the rat β2 subunit in that they contain all of the residues predicted to interact with the Mg-GTP substrate in the homodimeric receptor guanylyl cyclases (Liu et al, 1997).…”

“…Fig.·1B illustrates this high degree of identity between Gyc-88E and MsGC-β3. This alignment also includes Gyc-89Db, the rat β2 subunit, which displays NO-sensitive activity in the absence of an α subunit (Koglin et al, 2001), and a predicted MsGC-β3 orthologue from the Anopheles gambiae genome, CP12881. While the C-terminal extensions of Gyc-88E and MsGC-β3 are of similar lengths, they bear little similarity to each other except for two highly conserved stretches of 21 and 10 amino acids that contain several consensus phosphorylation sites.…”