Comparison of the properties of the five soluble guanylyl cyclase subunits in Drosophila melanogaster

Abstract

The Drosophila melanogaster genome contains 5 genes that code for soluble guanylyl cyclase subunits. Two of these genes code for subunits, Gycα-99B and Gycβ-100B, which form a conventional NO-sensitive guanylyl cyclase and the other three code for atypical subunits, Gyc-88E, Gyc-89Da and Gyc-89Db. The properties and distribution of Gyc-88E and Gyc-89Db have previously been described and here Gyc-89Da is described. Gyc-89Da only forms an active guanylyl cyclase when co-expressed with Gyc-88E. The three atypical subunits probably form two different heterodimers in vivo: Gyc-88E/89Da and Gyc-88E/89Db. Both of these heterodimers were slightly stimulated by NO donors and Gyc-88E/89Da showed a greater activation by Mn2+, with an increase in Vmax and a decrease in Km, compared to Gyc-88E/89Db. Both Gyc-88E/89Da and Gyc-88E/89Db were expressed in neurons in both the peripheral and central nervous system. Although all three heterodimeric soluble guanylyl cyclases in D. melanogaster can be activated by NO and inhibited by ODQ, the atypical enzymes can be distinguished from the conventional soluble guanylyl cyclase by their sensitivity to the NO-independent activators YC-1 and BAY 41-2272, which will only activate the conventional enzyme.Abbreviation:ORFopen reading frameUTRuntranslated region

Behavioral Responses to Hypoxia in Drosophila Larvae Are Mediated by Atypical Soluble Guanylyl Cyclases

“…Gyc-88E is the obligatory partner of Gyc-89Da and Gyc-89Db (Langlais et al 2004;Morton et al 2005). Since no lines with transposons in the Gyc-88E gene are currently available, we used RNA interference (RNAi) (Montgomery and Fire 1988) to reduce the levels of Gyc-88E in specific subsets of cells.…”

“…Gyc-89Da, Gyc-89Db, and Gyc-88E rescue fly lines: Gyc89Da ORF was amplified by PCR, cloned into pcDNA3.1 (Invitrogen, Carlsbad, CA) as described previously (Morton et al 2005), and then subcloned into the pUASt vector (https://dgrc.cgb.indiana.edu/) using the KpnI and XbaI restriction sites ( UAS Gyc-89Da). The Gyc-89Db ORF was obtained from the Berkeley Drosophila Genome Project (http:/ /www.…”

“…fruitfly.org/; clone ID GH09958), subcloned into pcDNA3.1, and then into the pUASt vector using the KpnI and XbaI restriction sites ( UAS Gyc-89Db). Gyc-88E ORF was amplified by PCR and cloned into pcDNA3.1, as described previously (Morton et al 2005), and then subcloned into the pUASt vector using the KpnI and NotI restriction sites ( UAS Gyc-88E). All constructs were confirmed by sequencing and injected into w 1118 embryos (http:/ /www.thebestgene.com/).…”

“…The Drosophila melanogaster genome contains five sGC subunit-encoding genes. As in mammalian systems, flies express the conventional a-and b-subunits (Gyca-99B and Gycb-100B) that function as obligate heterodimers and can be activated by nitric oxide (NO) (Morton et al 2005). In contrast, the remaining three sGC subunits, Gyc-88E, Gyc-89Da, and Gyc-89Db, belong to the atypical sGC family and have been shown to be regulated by oxygen (Morton 2004a).…”

“…The Gyc-89Da and Gyc-89Db subunits are up to 80% identical throughout their coding regions and are biochemically indistinguishable from each other, suggesting that they have arisen from a recent duplication (Morton et al 2005). Transient expression of different combinations of the Drosophila atypical sGCs in COS-7 cells showed that Gyc-89Da and Gyc-89Db form obligate heterodimers with Gyc-88E, and are potently activated in the absence of O 2 , therefore suggesting a function as O 2 sensors (Morton 2004b;Vermehren et al 2006;Morton and Vermehren 2007).…”