Receptor guanylyl cyclases in Inka cells targeted by eclosion hormone

Abstract

A signature of eclosion hormone (EH) action in insect ecdysis is elevation of cGMP in Inka cells, leading to massive release of ecdysis triggering hormone (ETH) and ecdysis initiation. Although this aspect of EH-induced signal transduction is well known, the receptor mediating this process has not been identified. Here, we describe a receptor guanylyl cyclase BdmGC-1 and its isoform BdmGC-1B in the Oriental fruit fly Bactrocera dorsalis that are activated by EH. The B form exhibits the conserved domains and putative N-glycosylation sites found in BdmGC-1, but possesses an additional 46-amino acid insertion in the extracellular domain and lacks the C-terminal tail of BdmGC-1. Combined immunolabeling and in situ hybridization reveal that BdmGC-1 is expressed in Inka cells. Heterologous expression of BdmGC-1 in HEK cells leads to robust increases in cGMP following exposure to low picomolar concentrations of EH. The B-isoform responds only to higher EH concentrations, suggesting different physiological roles of these cyclases. We propose that BdmGC-1 and BdmGC-1B are high- and low-affinity EH receptors, respectively.

Fig. 1.

Diagrammatic comparison of BdmGC-1 and -1B. Both BdmGC-1 isoforms exhibit all of the characteristics of receptor GCs, including (from left to right) an extracellular domain (ECD; 82–443 of BdmGC-1 and 82–489 of BdmGC-1B), a transmembrane region (TM; 504–524 of BdmGC-1 and 550–570 of BdmGC-1B), a kinase-homology domain (KHD; 584–845 of BdmGC-1 and 630–891 of BdmGC-1B), and a cyclase catalytic region (CYC; 911-1097 of BdmGC-1 957-1143 of BdmGC-1B). An extra insertion (347–392) with 4 additional cysteines in the ECD and absence of a C-terminal sequence are distinctive characteristics of BdmGC-1B.

Fig. 2.

BdmGC-1s are N-glycosylated in HEK-293T cells. Cells transiently expressing FLAG·BdmGC-1 or FLAG·BdmGC-1Β were lysed in buffer containing protease inhibitors and incubated in G7 reaction buffer in the presence or absence of PNgaseF for 30 min at 37 °C. Western blot analysis using an anti-FLAG M2 antibody was performed.

Fig. 3.

BdmGC-1 is expressed at main tracheal junctions. (A) Transcripts of BdmGC-1 and -1B expressed in third instar larvae and trachea. (B) RNA labeling with anti-sense RNA probes showing that BdmGC-1 transcripts are located at sites coincident with Inka cells at junctions along the main tracheal trunks. Specific Dig-labeled probes derived from the ECD region of BdmGC-1 were used for mRNA detection. Visualization is via alkaline phosphatase. (C) Inka cells exhibit strong ETH1-immunoreactivity, visualized with HRP-DAB. (D and E) BdmGC-1 signals visualized at junctions of tracheae using BdmGC-1 C-terminal-specific antibodies and BdmGC-1 N-terminal-specific antibodies, respectively, visualized by HRP/DAB. (F) N-terminal-specific antibodies neutralized by peptide antigen as control. (G) Merged view of fluorescent labeling of BdmGC-1 and propidium iodide counterstaining. (Scale bar, 20 μm.)

Fig. 4.

BdmGC-1 is colocalized with ETH1 and cGMP in epitracheal glands. Epitracheal glands from third instar larvae before pupal ecdysis were used for immuno-visualization. (A) Immuno-visualization of epitracheal gland by confocal microscopy. BdmGC-1 (red color; labeled by N-terminal-specific antibodies) shows cell surface expression and ETH-1 (green color) is localized in the cytoplasm of the Inka cell. The tissue is counterstained with DAPI (blue color). (B) To verify that Inka cells of B. dorsalis respond to EH by producing cGMP, tracheae of larvae were dissected and treated with 200 pM EH, then labeled with cGMP-specific antibodies and visualized by HRP/DAB. (C) Tracheae were treated with PBS instead of EH, followed by cGMP antibody labeling. (D and E) Epitracheal gland labeled by BdmGC-1 C-terminal specific (green color) antibodies and ETH1-(red color) antibodies, respectively. (F) Merged image of (E and F). Filled arrow: Inka cell, filled arrowhead: canal cell, open arrow: cell lying atop of epitracheal gland, and open arrowhead: cell adjacent to Inka cell. (Scale bars, 10 μm.)

Fig. 5.

HEK-293T cells transiently expressing BdmGC-1 or -1B generate cGMP in response to EH exposure. Cells transiently expressing FLAG·BdmGC-1 or FLAG·BdmGC-1B were incubated with serum-free medium for 1 h. Before assay, cells were washed twice with PBS and then incubated in PBS supplemented with IBMX for 10 min at room temperature. Thereafter, 200 pM EH was added and incubated for 30 min at 37 °C. Total cGMP content was measured as described in the Materials and Methods. GC activity was measured in HEK cells transiently transfected with empty vector as control. Results are expressed as means ± SEM (n = 3). Cell lysates were immunoblotted with anti-FLAG antibodies to confirm similar expression levels of BdmGC-1 and -1B form.

Fig. 6.

BdmGC-1 and -1B exhibit different sensitivities to EH. (A) HEK-293T cells transiently expressing FLAG·BdmGC-1 or FLAG·BdmGC-1B were incubated with increasing concentrations of EH, i.e., 64 fM, 320 fM, 1.6 pM, 8 pM, 40 pM, 200 pM, and 1 nM, for 30 min at 37 °C. Total cGMP mobilized was measured as described in the Materials and Methods and results are expressed as mean ± SEM (n = 3). Cell lysates were immunoblotted with anti-FLAG antibodies to confirm similar expression levels of BdmGC-1 and -1B. (B) Relative increase of cGMP production in response to physiological (8 pM) or pharmacological (1,000 pM) concentrations of EH for BdmGC-1 and -1B.

Fig. 7.

Model for EH-mediated ETH secretion by Inka cells. EH evokes cGMP synthesis by binding to its receptor, BdmGC-1. EH may act via 2 distinct pathways: A direct action on BdmGC-1 elevates cGMP content; a second signal transduction pathway functions via calcium mobilization, which regulates BdmGC-1B activity, a common regulation phenomenon in sensory GCs of mammals.