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First published online May 24, 2004
Journal of Experimental Biology 207, 2323-2338 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01025

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

Kristofor K. Langlais, Judith A. Stewart and David B. Morton^*

Departments of Integrative Biosciences and Cell and Developmental Biology, Oregon Health Sciences University, Portland, OR 97239, USA

View larger version (110K): [in a new window]   Fig. 1. Sequence analysis of Gyc-88E and Gyc-89Db. (A) Intron/exon structure and splice variants of the coding region of Gyc-88E. Exons are represented by boxes while introns are indicated with lines. The guanylyl cyclase functional domains are also indicated. The two splice variants are generated through the use of alternative splice/donor sites to vary how exons 10 and 11 are connected to yield Gyc-88E-S and Gyc-88E-L, which includes an additional 21 bp. The extra 21 bp in Gyc-88E-L translates into a seven amino acid stretch that contains potential PKC and PKG phosphorylation motifs, KITFS and KKIT, respectively. (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-ß3 (Nighorn et al., 1999), CP12881, the predicted orthologue of MsGC-ß3 in Anopheles gambiae (accession number EAA01162), and the rat ß2 subunit. Gyc-88E shares a high degree of sequence identity over the N-terminal and catalytic domains with MsGC-ß3 and CP12881, whereas the C-terminal domains are more divergent, except for two highly conserved sections of 21 and 10 amino acids (underlined). Gyc-88E, MsGC-ß3, CP12881 and the rat ß2 subunit all have the necessary catalytic residues (marked `B' for ß subunit residues and `A' for subunit residues) that are predicted to be required for forming an active homodimer (see Morton and Hudson, 2002 for a more extensive discussion). By contrast, Gyc-89Db has the residues characteristic of a ß subunit but is lacking some of those necessary for an subunit. All four of the insect subunits shown lack two cysteine residues (indicated with asterisks) required for NO activation, which are present in the rat ß2 subunit. A histidine residue, thought to be the axial ligand for the heme group in conventional /ß heterodimers (Zhao et al., 1998), is present in all of the subunits shown (indicated with a `+').

View larger version (12K): [in a new window]   Fig. 2. Phylogenetic tree showing the relationships between the ß and ß-like subunits of soluble guanylyl cyclases. The first atypical guanylyl cyclase subunit characterized, MsGC-ß3, clusters close to Gyc-88E, CP12881 from Anopheles and GCY-31 from C. elegans. A second grouping contains the remaining atypical subunits from Drosophila, Gyc-89Da, Gyc-89Db, P3998 from Anopheles, and GCY-33 from C. elegans. All the conventional soluble ß1 subunits, which form NO-sensitive 1/ß1 heterodimers, cluster together in a group that includes both vertebrate and invertebrate subunits. The remaining soluble guanylyl cyclases from C. elegans cluster together in a separate grouping and the mammalian ß2 subunits also appear to form a separate distinct cluster.

View larger version (41K): [in a new window]   Fig. 3. Expression of Gyc-88E in larvae and adults. (A) Northern blot showing the 6 kb transcript for Gyc-88E is present in both larvae (L) and adults (A). For a loading control, membranes were stripped and re-hybridized with a DIG-labeled riboprobe for the ribosomal protein RP49. (B) Both splice variants of Gyc-88E are expressed in both larvae and adults. RT-PCR was used to amplify across the junction between exons 10 and 11 to distinguish between Gyc-88E-S and Gyc-88E-L. Both a 70 bp (Gyc-88E-S) and a 91 bp band (Gyc-88E-L) were detected in samples from both larvae and adults in the presence of reverse transcriptase (+). These two bands were not observed when the reverse transcriptase was omitted (–). The 140 bp band observed in all lanes results from the amplification of Gyc-88E genomic DNA contamination.

View larger version (37K): [in a new window]   Fig. 4. Guanylyl cyclase activity of Gyc-88E and Gyc-89Db. COS-7 cells were transiently transfected with pcDNA3.1 vectors containing the open reading frames of various soluble guanylyl cyclase subunits and the cell extracts assayed for guanylyl cyclases activity under the conditions shown. (A) Gyc-88E exhibits enzyme activity in the absence of additional subunits and has higher levels of activity in the presence of Mn compared with Mg. The Manduca guanylyl cyclase, MsGC-ß3, exhibits similar properties and was included for comparison. The two splice variants of Gyc-88E (Gyc-88E-S and Gyc-88E-L) yielded similar levels of activity as each other. Data shown are the means ± S.E.M. of three determinations. (B) Kinetic analysis of Gyc-88E-S and Gyc-88E-L. Cell extracts were assayed for guanylyl cyclase activity in the presence of 0.1–10 mmol l-1 GTP in the presence of either 4 mmol l-1 Mg or Mn. A Michaelis–Menten curve was applied to the resulting data using Graphpad Prism 3.0. No difference in Km or Vmax was observed between the splice variants in the presence of Mg or Mn. (C) The NO donor sodium nitroprusside (SNP) stimulated the activity of both splice variants of Gyc-88E and this stimulation was unaffected by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxaline-1-one (ODQ). By contrast, ODQ virtually eliminated the activation of the Manduca MsGC-1/ß1 heterodimer by SNP. Assays were carried out in the presence of 4 mmol l-1 Mg. Data shown are the means ± S.E.M. of three determinations. (D) Guanylyl cyclase activity of Gyc-89Db. No enzyme activity was detected when Gyc-89Db was expressed in the absence of additional subunits or when co-expressed with Gyc-99B in either the presence of 4 mmol l-1 Mg or 4 mmol l-1 Mn. However, when Gyc-89Db was co-expressed with Gyc-88E, greater basal activity was detected than when Gyc-88E was expressed alone. The basal activity was enhanced in the presence of both Mg and Mn. The data shown represent pooled values for Gyc-88E-S and Gyc-88-L, as no differences were seen between the different splice variants. Data shown represent the means ± S.E.M. of six determinations. (E–G) Guanylyl cyclase activity of the Drosophila soluble guanylyl cyclase subunits in the presence of 100 µmol l-1 of the NO donors SNP, SIN-1, SNAP, SNOG, DEA-NONOate, NOC-12 and spermine NONOate and the NO-independent activator of soluble guanylyl cyclase YC-1. The subunit combinations shown are Gyc-88E (E), Gyc-88E co-expressed with Gyc-89Db (F) and Gyc-99B/Gycß-100B (G). The data shown represent pooled values for Gyc-88E-S and Gyc-88-L, as no differences were seen between the different splice variants. Data shown represent the means ± S.E.M. of at least four determinations. For all graphs, the data were analyzed using one-way ANOVA: `ns' represents P>0.05, **P<0.01 and ***P<0.001. For the data shown in A, C and D, Tukey–Kramer post-hoc test was used and, for E–G, Dunnett's multiple comparison test was used.

View larger version (84K): [in a new window]   Fig. 5. Localization of Gyc-88E and Gyc-89Db expression in the central nervous system of Drosophila embryos. In situ hybridization experiments were performed on whole embryos using fragmented DIG-labeled Gyc-88E or Gyc-89Db riboprobes. (A,C) Gyc-88E expression in stage 16 embryos. Expression was detected in a segmental pattern in the ventral nerve cord and throughout the brain (open arrowhead). (B,D) Gyc-89Db expression in stage 17 embryos. A similar pattern of expression was detected in the ventral nerve cord and brain, although noticeably fewer cells stain in the brain with Gyc-89Db (open arrowhead). A and B show the horizontal view, while C and D show the lateral view. Application of a sense probe generated from Gyc-88E (E) or Gyc-89Db (F) demonstrates that the low level of ubiquitous background staining observed reflects non-specific hybridization. Anterior is always left and ventral is down in side views. Square brackets in C and D indicate the position of the ventral nerve cord. Scale bar, 100 µm.

View larger version (85K): [in a new window]   Fig. 6. Gyc-88E and Gyc-89Db expression in the central nervous system of third instar larvae. In situ hybridization was performed on isolated third-instar central nervous systems using the same riboprobes used in the embryo experiments. (A,B) Expression in the brain lobes. Gyc-88E (A) and Gyc-89Db (B) expression was found in scattered cells throughout the brain but was most prominent in a small cluster of cells located in the anterior medial region of each brain lobe (open arrowheads). (C–F) Expression in the ventral nerve cord. Gyc-88E (C,E) and Gyc-89Db (D,F) expression was found in scattered cells located laterally and in the midline (midline marked with broken line). The pair of images shown in C and E and in D and F are from the same preparation but are viewed at different focal planes. Anterior is up in all panels. Scale bar, 200 µm.

View larger version (130K): [in a new window]   Fig. 7. Localization of Gyc-88E and Gyc-89Db in cells associated with the embryonic peripheral nervous system. In situ hybridization on stage 17 embryos also revealed that Gyc-88E or Gyc-89Db were both expressed in several peripherally located cells. (A,C) Expression of Gyc-88E. (B,D) Expression of Gyc-89Db. A and B show the horizontal view, while C and D show the lateral view. On each side of the embryo, two cells were detected in segments T1, T2 and T3, arranged in either an upper row or a lower row of cells, and a single cell was detected in A1 and A2. Left is always anterior and down is ventral in side views. (E) Application of both probes simultaneously resulted in the same number of cells staining in segments T1–A2 as when each probe was applied individually. (F,G) Expression of Gyc-88E and Gyc-89Db in the head segment. Gyc-88E (F) was expressed in a pair of cells, while Gyc-89Db (G) was expressed in more cells (2–5 per side in the clusters in focus, and 4–5 per side in clusters located in a more posterior position and out of focus, indicated with open arrowheads). Horizontal views are shown, and anterior is up. (H–J) Expression of Gyc-88E and Gyc-89Db in segments A8 and A9. Three consecutive focal planes of focus are shown starting from most ventral (left) to dorsal (right) to capture all cells. Both Gyc-88E (H) and Gyc-89Db (I) are expressed in a total of 12 cells. The cells are numbered arbitrarily. (J) When both probes were used simultaneously, the number of cells detected remained the same. Scale bar, 200 µm.

View larger version (111K): [in a new window]   Fig. 8. Gyc-88E and Gyc-89Db are expressed in neurons of the peripheral nervous system. In situ hybridization (blue/black stain) was combined with immunohistochemical staining with the neuronal antibody 22C10 (brown stain). Stained cells are indicated with open arrowheads and are identified where possible. (A,B) Expression of Gyc-88E and Gyc-89Db in the lateral neuron clusters of segments T2 and T3. The example shown is segment T3 (T2 was identical). Gyc-88E (A) and Gyc-89Db (B) were expressed in one of the three les neurons that innervate a lateral external basiconical sensillum. In both of these panels, dendrites from the three les neurons can be observed to extend towards the basiconical sensillum (location marked with an asterisk). (C,D) Expression of Gyc-88E and Gyc-89Db in the ventral neuron clusters of segments T2 and T3. Gyc-88E (C) and Gyc-89Db (D) were expressed in one of the three ves neurons that innervate a ventral external basiconical sensillum. Again, the example shown is in segment T3 (T2 was identical). (E,F) Expression of Gyc-88E and Gyc-89Db in the neuron clusters of segments A1 and A2. Gyc-88E (E) and Gyc-89Db (F) were expressed in one of two v'td that innervate specific tracheal branches. The example shown is in segment A1 (A2 was identical but the staining was less intense). The stained cell was always the most anterior of the pair. (G–I) Expression of Gyc-88E and Gyc-89Db in the head segment. Gyc-89Db was expressed in 4–5 neurons in each of the dorsal ganglia (one on each side) (G), and in 2–5 neurons in each of the terminal (maxillary) ganglia (one on each side) (H). Gyc-88E expression was found in one neuron in each terminal ganglion (I). Note the dendrites projecting towards the head sensilla. (J,K) Expression of Gyc-88E and Gyc-89Db in the caudal sensory cones in the telson. Gyc-88E (J) and Gyc-89Db (K) were expressed in one of at least two neurons that innervate each of the caudal sensory cones in segment A9. The neighboring neuron that does not express guanylyl cyclase is indicated with an asterisk. In K, the dendrite is clearly seen to extend from the neuron that expressed Gyc-89Db to the extreme tip of the sensory cone, appearing to extend past the edge of the main body of the cone as a short protrusion. Scale bar, 30 µm.

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