1Department of Entomology, Texas A&M University (TAMU), 2Department of Molecular and Cellular Medicine, Texas A&M University (TAMU)
Lu, H., Kersch, C. N., Taneja-Bageshwar, S., Pietrantonio, P. V. A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors. J. Vis. Exp. (50), e2732, doi:10.3791/2732 (2011).
Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.
1. Establishment of stable cell lines
2. The calcium bioluminescence plate assay
3. Instrument operation and data analysis
4. Representative Results:
When expressed in CHO-K1 cells, the mosquito Aedes aegypti kinin receptor behaved as a multiligand receptor and functionally responded to concentrations as low as 1 nM of the three endogenours Aedes kinins, Aedae kinins 1-3, tested singly using the calcium bioluminescence plate assay. Figure 1 shows that the rank order of potency obtained was Aedae-K-3 > Aedae-K-2 > Aedae-K-1, based on the respective EC50 values of Aedae-K-3, 16.04 nM; Aedae-K-2, 26.6 nM and Aedae-K-1, 48.85 nM, which were statistically significantly different (P < 0.05) (Pietrantonio et al., 2005).
We also used this assay to determine which kinin residues are critical for the kinin peptide-receptor interaction. Insect kinin peptides share a C-terminal pentapeptide that represents the minimal sequence required for biological activity, also known as core. In Table 1, the kinin peptide core analogs were synthesized as an Alanine replacement series of the core kinin pentapeptide FFSWGa and tested by a calcium bioluminescence plate assay (Taneja-Bageshwar et al., 2006). We found that the amino acids Phe1 and Trp4 were essential for activity of the insect kinins for both receptors.
The assay also can be used to test peptides designed for enhanced biostability. Table 2 shows the designed kinin analogs containing amino isobutyric acid (Aib) tested on the tick recombinant kinin receptor and Figure 2 shows the activity comparison of six alpha-amino isobutyric acid analogs on the tick kinin receptor expressing CHO-K1 cell line by a calcium bioluminescence plate assay (Taneja-Bageshwar et al., 2009). The hexapeptide analog FFFSWGa is added for a positive control for receptor activity. The analog FF[Aib]WGa resulted more active than this hexapeptide control analog. The analog with two aminoisobutyric acid replacements, [Aib]FF[Aib]WGa, was the most potent of the double-replacement analogs tested (Table 2 and Figure 2).
For more examples of how this assay has been and can be applied see Nachman and Pietantonio (2010), Nachman et al. (2009), Taneja-Bageshwar et al. (2008a), and Taneja-Bageshwar et al. (2008b).
Figure 1. Estimation of Aedes kinins effective concentration (EC50) on CHO-K1 E10 cells by a calcium-bioluminescence plate assay. The y-axis in the concentration-response curves was obtained from bioluminescence units expressed as a percentage of the maximal response observed for each peptide. Data points represent the average of six replicates obtained during three independent experiments. Bars represent the standard error. (A) Estimation of Aedae-K1 EC50 = 48 nM. (B) Estimation of Aedae-K2 EC50 = 26 nM. (C) Estimation of Aedae-K3 EC50 = 16 nM. EC50 Aedae-K3 < EC50 Aedae-K2 < EC50 Aedae-K1; P < 0.05. Statistical analysis and graphs were with the GraphPad Prism 4.0 software.
Figure 2. Activity comparison of six alpha-amino isobutyric acid analogs on the tick kinin receptor expressing CHO-K1 cell line by a calcium bioluminescence plate assay. The y-axis represents percent maximal bioluminescence units for each analog expressed as a percentage of bioluminescence observed at a concentration versus the maximal response observed among all concentrations tested for each analog. Statistical analysis and graphs were performed with GraphPad Prism 4.0 software.
|Tick receptor cell line||Mosquito receptor cell line|
|Peptides||EC50 (nM)||Maximal bioluminescence response at 1 mM||EC50 (nM)||Maximal bioluminescence response at 1 mM|
Table 1. Estimated potencies (EC50) and maximal bioluminescence response of all the peptides tested on tick and mosquito receptor transfected cell lines*.
*The EC50 estimates the concentration required to induce a half-maximal response. I: Inactive if bioluminescence response is less than 300 units (level of vector-only transfected cells). A: The position where the respective residue in the peptide FFSWGa has been replaced by alanine. N.D.: The analog was tested but was either not very active or was not active at lower molarities, thus an EC50 could not be determined.
Table 2. Kinin analogs (K) containing amino isobutyric acid (Aib) tested on the tick recombinant kinin receptor. Ac: acetyl; α Me: α methyl- phenylalanine; β3F: β3 -phenylalanine; a: amide.
We were able to perform the functional characterization of the first neuropeptide receptor discovered from the Arachnida (ticks, mites and spiders), the tick kinin receptor, using this protocol. This method has three primary applications. First, the technique can be applied for receptor deorphanization through ligand activity measurements. Second, the assay can resolve ligand-receptor structure-activity relationships (SAR). Third, the methods can be used in drug discovery. Furthermore, this protocol can be used to study the activity of agonists or antagonists on almost any GPCR. We are beginning to adapt this protocol for screening of small libraries. The cell line we utilized does not express the ubiquitous G protein G16. We did not need it because arthropod kinin receptors signal through the Gq protein and the intracellular calcium cascade and they conserve this signaling properties in mammalian cells as shown here.
No conflicts of interest declared.
Drs. C. J. P. Grimmelikhuijzen and Michael Williamson from University of Copenhagen (Denmark) are appreciated for providing the aequorin plasmid. Our collaborator, Dr. Ronald J. Nachman from ARS-USDA (TX, USA), is acknowledged for peptide synthesis and for providing the NOVOstar plate reader.
|CHO-K1 cells||ATCC||CCL-61||Manassas, VA, USA|
|Fetal Bovine Serum||Sigma-Aldrich||F0643|
|Opti-MEM I Reduced Serum Medium||Invitrogen||31985|
|QIAprep spin miniprep kit||Qiagen||19064|
|FuGENE 6 Transfection reagent||Roche Group||11 814 443 001|
|96-well white thin bottom microtitere plate||Costar||3610|
|calcium-free DMEM media||Invitrogen||21068|
|Bright-Line Hemacytometer||Hausser Scientific||Horsham, PA|
|Prism software 4.0||GraphPad Software Inc.||San Diego, CA, USA|
|T-25 and T-75 Flasks||BD Biosciences||353014 and 353135|