Identification and Characterization of a Novel β Subunit of Soluble Guanylyl Cyclase That Is Active in the Absence of a Second Subunit and Is Relatively Insensitive to Nitric Oxide

Abstract

Previously characterized soluble guanylyl cyclases form ␣-␤ heterodimers that can be activated by the gaseous messenger, nitric oxide. In mammals, four subunits have been cloned, named ␣1, ␣2, ␤1, and ␤2. We have identified a novel soluble guanylyl cyclase isoform from the nervous system of the insect Manduca sexta that we have named M. sexta guanylyl cyclase ␤3 (MsGC-␤3). It is most closely related to the mammalian ␤ subunits but has several features that distinguish it from previously identified soluble cyclases. Most importantly, MsGC-␤3 does not need to form heterodimers to form an active enzyme because guanylyl cyclase activity can be measured when it is expressed alone in COS-7 cells. Moreover, this activity is only weakly enhanced in the presence of the nitric oxide donor, sodium nitroprusside. Several of the amino acids in rat ␤1 subunits, previously identified as being important in heme binding or necessary for nitric oxide activation, are substituted with nonsimilar amino acids in MsGC-␤3. There are also an additional 315 amino acids C-terminal to the catalytic domain of MsGC-␤3 that have no sequence similarity to any known protein. Northern blot analysis shows that MsGC-␤3 is primarily expressed in the nervous system of Manduca.The intracellular messenger cGMP mediates a wide variety of cellular and physiological processes. It functions as the primary messenger in visual transduction (1, 2), is an important regulator in vascular smooth muscle and kidney function (3, 4), and has been implicated in a number of forms of neuronal plasticity (5-7).The enzymes that regulate the synthesis of cGMP fall into two major classes: the cytoplasmically localized, soluble guanylyl cyclases and the membrane associated, receptor guanylyl cyclases (4). Soluble guanylyl cyclases are obligate heterodimers composed of an ␣ and a ␤ subunit. Two ␣ (␣1 and ␣2) and two ␤ (␤1 and ␤2) subunits have been cloned from mammalian tissues (8 -13). The active heterodimer requires and binds heme as a prosthetic group and can be potently activated by the gaseous messenger, nitric oxide (NO) 1 (14). The soluble cyclases are found in the cytosol of cells, although the mammalian ␤2 subunit has a consensus isoprenylation site, suggesting that it might be associated with membranes. The receptor guanylyl cyclases, by contrast, are integral membrane proteins with transmembrane and extracellular domains (4).We have been using the insect Manduca sexta as a model system to study cGMP regulation and have previously shown that a neuropeptide, eclosion hormone, is a potent stimulator of cGMP levels in the central nervous system (15). Biochemical characterization of the eclosion hormone-stimulated cGMP increase suggested that eclosion hormone might activate a NOinsensitive, soluble guanylyl cyclase (15-18). In an attempt to identify the pathway for eclosion hormone-stimulated cGMP levels, we used reverse transcription-polymerase chain reaction (RT-PCR) to identify guanylyl cyclases from Manduca nervous tissue. This approach has yielded a number of dif...

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

“…Unlike all known β1 subunits and the mammalian β2 subunits, both Gyc-88E and MsGC-β3 have substitutions at two cysteine residues known to be crucial for heme binding and NO activation in the rat β1 subunit (Friebe et al, 1997;Fig.·1B). Extracts made from COS-7 cells transiently transfected with MsGC-β3 yielded no increase in activity over basal levels when NO donors were applied (Nighorn et al, 1999). This observation, together with its sequence features, led to the prediction that Gyc-88E would also be NO-insensitive (Morton and Hudson, 2002).…”

“…Gyc-88E exhibited similar properties, with both slice variants yielding significantly higher levels of activity in the presence of Mn compared with Mg (Fig.·4A). As a comparison, we also transfected COS-7 cells with a plasmid coding for the Manduca guanylyl cyclase, MsGC-β3 (Nighorn et al, 1999). The activity of MsGC-β3 in the presence of either Mg or Mn was at least 10-fold higher than that of Gyc-88E (Fig.·4A), but whether this was due to an intrinsically higher level of specific activity or whether it reflected higher levels of protein expression is not known.…”

“…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.…”

“…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).…”

“…(B) Multiple sequence alignment of Gyc-88E and Gyc-89Db with selected atypical guanylyl cyclase subunits. The other sequences included in the alignment are MsGC-β3 (Nighorn et al, 1999), CP12881, the predicted orthologue of MsGC-β3 in Anopheles gambiae (accession number EAA01162), and the rat β2 subunit. Gyc-88E shares a high degree of sequence identity over the N-terminal and catalytic domains with MsGC-β3 and CP12881, whereas the C-terminal domains are more divergent, except for two highly conserved sections of 21 and 10 amino acids (underlined).…”