MsGC-β3 forms active homodimers and inactive heterodimers with NO-sensitive soluble guanylyl cyclase subunits

Abstract

SUMMARY Soluble guanylyl cyclases are typically obligate heterodimers, composed of a single alpha and a single beta subunit. MsGC-β3, identified in the tobacco hornworm Manduca sexta, was the first example of a soluble guanylyl cyclase that exhibited enzyme activity without the need for coexpression with additional subunits. Subsequent studies have revealed that the mammalian β2 subunit also shares this property. Using a combination of gel filtration chromatography, coprecipitation and site-directed mutagenesis we show that, as predicted, MsGC-β3 forms active homodimers. We also demonstrate that MsGC-β3 is capable of forming heterodimers with the nitric oxide (NO)-sensitive guanylyl cyclase subunits MsGC-α1 and MsGC-β1. These heterodimers, however, show no enzyme activity and, like mammalian β2 subunits, act in a dominant negative manner when combined with the NO-sensitive subunits to disrupt their activation by NO. In addition,we show that the unique C-terminal domain of MsGC-β3 is not necessary for enzyme activity and might act as an auto-inhibitory domain.

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

“…The C-terminal extensions of MsGC-β3, Gyc-88E and the Anopheles orthologue CP12881 are highly divergent, except for two conserved stretches of 21 and 10 amino acids, suggesting that these regions play an important role in enzymatic regulation. Removing the entire C-terminal extension from MsGC-β3 decreased the Km in the presence of Mg, while no change was measured in the presence of Mn (Morton and Anderson, 2003). These results suggested that the C-terminal domain formed an auto-inhibitory domain in MsGC-β3 (Morton and Anderson, 2003).…”

“…Removing the entire C-terminal extension from MsGC-β3 decreased the Km in the presence of Mg, while no change was measured in the presence of Mn (Morton and Anderson, 2003). These results suggested that the C-terminal domain formed an auto-inhibitory domain in MsGC-β3 (Morton and Anderson, 2003). The estimated values for Km for Gyc-88E were similar to those for MsGC-β3, in particular the almost 20-fold reduction in the presence of Mn compared with Mg, suggesting that the C-terminal domain of Gyc-88E might also be inhibitory.…”

“…Firstly, like the receptor guanylyl cyclases and unlike all known β1 subunits, both Gyc-88E and MsGC-β3 possess all of the residues thought to interact with the Mg-GTP substrate ( Fig.·1B; for a detailed discussion of homodimer/heterodimer predictions and a model of the catalytic site, see Morton and Hudson, 2002). Previous studies showed that MsGC-β3 does yield basal activity in the absence of other subunits (Nighorn et al, 1999) and forms homodimers (Morton and Anderson, 2003). These studies formed the basis for the prediction that Gyc-88E would also yield basal activity in the absence of other subunits, a prediction demonstrated to be correct in this and a previous study ( Fig.·4; Morton, 2004).…”

“…A cDNA of Gyc-89Db that contained the full ORF was obtained as an expressed sequence tag (EST) cDNA (clone ID: GH09958) from the Berkeley Drosophila Genome Project and subcloned into pcDNA3.1 utilizing EcoRI restriction enzyme sites. COS-7 cells were transiently transfected with constructs and the homogenates assayed for guanylyl cyclase activity as described previously (Morton and Anderson, 2003). 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) was dissolved in dimethyl sulfoxide (DMSO; Sigma, St Louis.…”

“…Gycα-99B and Gycβ-100B are the orthologues of the conventional mammalian α1 and β1 subunits, which form obligate α/β heterodimers that are potently activated by the gaseous messenger molecule NO (Lucas et al, 2000). One of the remaining three, Gyc-88E, is the orthologue of MsGC-β3, a Manduca sexta soluble guanylyl cyclase subunit that forms active homodimers and is insensitive to NO (Nighorn et al, 1999;Morton and Anderson, 2003). Additional orthologues of MsGC-β3 include CP12881, a predicted subunit identified in the A. gambiae genome, and GCY-31, identified in C. elegans.…”