Identification, Functional Characterization, and Pharmacological Analysis of Two Sulfakinin Receptors in the Medically-Important Insect Rhodnius prolixus

Abstract

The chordate gastrin/cholecystokinin and ecdysozoan sulfakinin (SK)-signaling systems are functionally and structurally homologous. In the present study, we isolated the cDNA sequences encoding the SK receptors in Rhodnius prolixus (Rhopr-SKR-1 and Rhopr-SKR-2). The Rhopr-SKRs have been functionally characterized and their intracellular signaling pathways analysed via a functional receptor assay. Both Rhopr-SKRs are exclusively activated via the two native R. prolixus sulfakinins, Rhopr-SK-1 and Rhopr-SK-2, but not via nonsulfated Rhopr-SK-1. The Rhopr-SKRs are each linked to the intracellular Ca²⁺ second messenger pathway, and not to the cyclic AMP pathway. Spatial transcript expression analyses revealed that each Rhopr-SKR is predominantly expressed in the central nervous system with lower expression throughout peripheral tissues. The critical importance of the SK-signaling pathway in the blood-feeding behaviour of R. prolixus was demonstrated by knockdown of the transcripts for Rhopr-SKs and Rhopr-SKRs, which results in an increase in the mass of blood meal taken. The parasite causing Chagas disease is transmitted to the host after R. prolixus has taken a blood meal, and characterization of the SKRs provides further understanding of the coordination of feeding and satiation, and ultimately the transmission of the parasite.

Figure 1

Rhodnius prolixus cDNA sequences and the corresponding amino acid sequences of (A) Rhopr-SKR-1 and (B) Rhopr-SKR-2. The numbers of the nucleotides and amino acids (italicized) are to the right of the sequences. For each receptor, the bolded and highlighted methionine is the translation start site. The predicted N-linked glycosylation sites are boxed. The seven predicted hydrophobic transmembrane domains are bolded, highlighted, and underlined. Predicted phosphorylation sites are shaded in grey. Exon boundaries are double-underlined. The ERY and NPITY motifs are italicized and boxed. The 3′ poly(A) tails are underlined.

Figure 2

Functional characterization of Rhopr-SKR-1 transiently expressed in HEK293/CNG cells. (A) Dose-response curve showing the activation of Rhopr-SKR-1 via Rhopr-SK-1, Rhopr-SK-2, and ns (nonsulfated) Rhopr-SK-1 as ligands (for the first 5-second interval). EC₅₀ for Rhopr-SK-1 was 5.32 × 10⁻¹⁰ M, whilst the EC₅₀ for Rhopr-SK-2 was 8.69 × 10⁻⁹ M. Nonsulfated Rhopr-SK-1 was unable to elicit a response. (B) Kinetics of response activation at 5-second intervals (a total of 15 seconds) at 10⁻⁶ M for Rhopr-SK-1 and Rhopr-SK-2. (C) Kinin-2 (Rhopr-Kinin-2), tachykinin (Rhopr-TK-2), and GNDNFMRFamide showed little to no response. Data points are mean ± standard error of the mean for 3 replicates.

Figure 3

Functional characterization of Rhopr-SKR-2 transiently expressed in HEK293/CNG cells. (A) Dose-response curve showing the activation of Rhopr-SKR-2 via Rhopr-SK-1 and Rhopr-SK-2, and ns (nonsulfated) Rhopr-SK-1 as ligands (for the first 5-second interval). EC₅₀ for Rhopr-SK-1 was equivalent to 1.44 × 10⁻¹⁰ M, whilst the EC₅₀ for Rhopr-SK-2 was 1.01 × 10⁻¹⁰ M. Nonsulfated Rhopr-SK-1 was unable to elicit a response. (B) Kinetics of response activation at 5-second intervals (a total of 15 seconds) at 10⁻⁶ M for Rhopr-SK-1 and Rhopr-SK-2. (C) Kinin-2 (Rhopr-Kinin-2), tachykinin (Rhopr-TK-2), and GNDNFMRFamide showed little to no response. Data points are mean ± standard error of the mean for 3 replicates.

Figure 4

Luminescence response of Rhopr-SK-1 (1 × 10⁻⁶ M), Rhopr-SK-2 (1 × 10⁻⁶ M), as well as a mixture of Rhopr-SK-1 and Rhopr-SK-2 (1 × 10⁻⁶ M) for (A) Rhopr-SKR-1 and (B) Rhopr-SKR-2. For Rhopr-SKR-1, the observed luminescence was significantly lower for the Rhopr-SK-1 and Rhopr-SK-2 mixture as opposed to Rhopr-SK-1 alone (one way ANOVA followed by Tukey’s Multiple Comparisons Test, ****P < 0.0001). For Rhopr-SKR-2, luminescence was lower for the combination of Rhopr-SK-1 and Rhopr-SK-2 as opposed to Rhopr-SK-1 alone (one way ANOVA followed by Tukey’s Multiple Comparisons Test, P < 0.05), and as opposed to Rhopr-SK-2 alone (one way ANOVA followed by Tukey’s Multiple Comparisons Test, **P < 0.01). Here, luminescence was lower for Rhopr-SK-2 when compared to Rhopr-SK-1 (one way ANOVA followed by Tukey’s Multiple Comparisons Test, ***P < 0.001). Both receptors were transiently expressed in HEK293/CNG cells. Data points are mean ± standard error of the mean for 3 replicates.

Figure 5

Functional characterizations of Rhopr-SKRs in the presence or absence of the PLC inhibitor, U73122 or in calcium-free media containing EGTA. Namely, (A) Rhopr-SKR-1 with U73122, (B) Rhopr-SKR-2 with U73122, (C) Rhopr-SKR-1 with EGTA, and (D) Rhopr-SKR-2 with EGTA. All receptors were transiently expressed in HEK293/CNG cells, with all media containing calcium (in the absence of EGTA). For both Rhopr-SKR-1 and Rhopr-SKR-2, both Rhopr-SK-1 and Rhopr-SK-2 were unable to elicit a response in the presence of 10 μM of U73122 when compared to Rhopr-SK-1 and Rhopr-SK-2 in the absence of U73122. In the absence of U73122 for Rhopr-SKR-1, EC₅₀ for Rhopr-SK-1 was 3.17 × 10⁻¹⁰ M and 1.33 × 10⁻⁸ M for Rhopr-SK-2. For Rhopr-SKR-2, EC₅₀ for Rhopr-SK-1 was 1.03 × 10⁻¹⁰ M and 1.21 × 10⁻¹⁰ M for Rhopr-SK-2. In the absence of EGTA for Rhopr-SKR-1, EC₅₀ for Rhopr-SK-1 was 2.94 × 10⁻¹⁰ M and 6.86 × 10⁻⁹ M for Rhopr-SK-2. In the presence of EGTA for Rhopr-SKR-1, EC₅₀ for Rhopr-SK-1 was 4.40 × 10⁻¹⁰ M and 3.02 × 10⁻⁸ M for Rhopr-SK-2. In the absence of EGTA for Rhopr-SKR-2, EC₅₀ for Rhopr-SK-1 was 2.14 × 10⁻¹⁰ M and 1.34 × 10⁻¹⁰ M for Rhopr-SK-2. In the presence of EGTA for Rhopr-SKR-2, EC₅₀ for Rhopr-SK-1 was 3.44 × 10⁻¹⁰ M and 3.14 × 10⁻¹⁰ M for Rhopr-SK-2. Data points are mean ± standard error of the mean for 3 replicates.

Figure 6

Spatial expressions of the (A) Rhopr-SKR-1 and (B) Rhopr-SKR-2 transcripts in Rhodnius prolixus 5^th instars via reverse transcriptase quantitative PCR (RT-qPCR). For each receptor, expression was examined in the CNS, foregut, anterior midgut, posterior midgut, hindgut, salivary glands, heart, Malpighian tubules, fat body, male reproductive system, and female reproductive system. Data points are mean ± standard error of the mean for 3 independent samples.

Figure 7

Verification of Rhopr-SK knockdown via immunohistochemical staining of the brain in 5th instar Rhodnius prolixus. Insects were injected with the dsRNA of either (A) ARG (dsARG) or (B) Rhopr-SK (dsRhopr-SK) then dissected and examined two days post-injection. Animals injected with dsARG display bright immunoreactive staining throughout the cells and processes of the brain, whereas Rhopr-SK dsRNA-injected animals display reduced staining throughout the same regions. Scale bars: 100 μm.

Figure 8

Post-feeding weights of 5^th instar Rhodnius prolixus previously injected (48 hours earlier) with (A) 1 μg of Rhopr-SK dsRNA (dsRhopr-SK) or 1 μg of ARG dsRNA (dsARG); or with (B) a mixture of 1 μg of Rhopr-SKR-1 and 1 μg of Rhopr-SKR-2 dsRNA (dsRhopr-SKRs) or 2 μg of ARG dsRNA (dsARG). Post-feeding weights were significantly lower in control insects injected with ARG dsRNA in comparison to insects injected with either Rhopr-SK dsRNA (two way ANOVA followed by Sidak’s Multiple Comparisons Test, ****P < 0.0001) or Rhopr-SKR-1 with Rhopr-SKR-2 dsRNA (two way ANOVA followed by Sidak’s Multiple Comparisons Test, ****P < 0.0001). Histograms are mean ± standard error of the mean for 25 insects (for each of A,B).