Identification of RL-TGR, a coreceptor involved in aversive chemical signaling

Abstract

Chemical signaling plays an important role in predator-prey interactions and feeding dynamics. Like other organisms that are sessile or slow moving, some marine sponges contain aversive compounds that defend these organisms from predation. We sought to identify and characterize a fish chemoreceptor that detects one of these compounds. Using expression cloning in Xenopus oocytes coexpressing the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, the beta-2 adrenergic receptor (beta(2)AR), and fractions of a zebrafish cDNA library, we isolated a cDNA clone encoding receptor activity-modifying protein (RAMP)-like triterpene glycoside receptor (RL-TGR), a novel coreceptor involved in signaling in response to triterpene glycosides. This coreceptor appears to be structurally and functionally related to RAMPs, a family of coreceptors that physically associate with and modify the activity of G protein-coupled receptors (GPCRs). In membranes from formoside-responsive oocytes, RL-TGR was immunoprecipitated in an apparent complex with beta(2)AR. In HEK293 cells, coexpression of beta(2)AR induced the trafficking of RL-TGR from the cytoplasm to the plasma membrane. These results suggest that RL-TGR in the predatory fish physically associates with the beta(2)AR or another, more physiologically relevant GPCR and modifies its pharmacology to respond to triterpene glycosides found in sponges that serve as a potential food source for the fish. RL-TGR forms a coreceptor that responds to a chemical defense compound in the marine environment, and its discovery might lead the way to the identification of other receptors that mediate chemical defense signaling.

Fig. 1.

Identification and initial characterization of clone encoding a coreceptor for aversive compounds. (A and B) Traces in response to 1 μM isoproterenol (I) or 5 μM formoside (F) from oocytes coexpressing CFTR, β₂AR, and either cDNA library fraction A (n = 10; formoside response range, 0.05–0.2 μA) (A) or isolated full-length clone A9-f4-230 (n > 40; range, 0.1–3.7 μA) (B) (Scale bars: A, 0.5 μA, 2 min; A, Inset, 0.2 μA, 2 min; B, 0.5 μA, 2 min.) The inset in A shows a view of the same trace expanded in the amplitude dimension. (C and D) Electrophysiological responses to various compounds in oocytes expressing CFTR, β₂AR, and the full-length clone. (C) A mixture of ectyoplasides A and B (E) caused a response comparable to a formoside-induced response (n = 5; range, 0.1–0.4 μA) (Scale bar: 0.5 μA, 1 min.) (D) The ligand specificity of RL-TGR was investigated by exposing formoside-responsive oocytes to a variety of other compounds, including ectyoplasides A and B (10 μM), ouabain (5 μM), digoxin (5 μM), 17β-estradiol (5 μM), octanal (0.5 mM), cycloheximide (1.5 μM), and capsaicin (50 μM). Responses to these ligands were normalized to the response to 5 μM formoside in the same cell (n = 3–10). Error bars represent SD. *P < 0.05 compared with formoside in the same cell. (E) RT-PCR analysis of zebrafish tissues identified transcript in the head (H) and trunk (T). (F) RT-PCR analysis of bluehead wrasse head tissue.

Fig. 2.

RL-TGR is responsible for the formoside-induced response. The largest ORF from the active clone was expressed as a Strep II/His–tagged fusion protein. (A) Representative response after the application of 1 μM isoproterenol (I) and 5 μM formoside (F) to oocytes expressing CFTR, β₂AR, and Strep II (S)/His (H)–tagged receptor fusion protein (see Inset) (n = 19; range, 0.05–2.2 μA). (Scale bar: 0.4 μA, 2 min.) (B) Western blot analysis showing Strep II/His–tagged receptor in formoside-responsive oocytes also expressing CFTR and β₂AR, immunoprecipitated with anti-His antibody and immunoblotted against Strep II. (C) Predicted peptide sequence of RL-TGR. Filled boxes denote extracellular cysteines. The predicted transmembrane region is underlined. Bracketed residues are encoded by the conserved DNA segment. (D) Transmembrane prediction plot (Left) and schematic of RL-TGR (Right). Red indicates the probability of being intracellular, blue indicates the probability of being extracellular, and gray bars represent transmembrane probability. Black-filled circles in (C) and (D) represent the putative PDZ-binding domain. (E) Confirmation of membrane topology. RL-TGR was expressed in HEK293 cells with a C-terminal His-tag (RL-TGR–His). Except where noted, immunofluorescence was performed in nonpermeabilized cells. (Panel 1) Cells expressing RL-TGR–His, probed for His tag. (Panel 2) Cells expressing RL-TGR–His, probed for actin. (Panel 3) To visualize actin, cells expressing RL-TGR–His were permeabilized before immunostaining for actin. (Panel 4) Cells transfected with empty vector, probed for His tag. (Panel 5) Cells expressing RL-TGR–His, probed with secondary antibody only. In each panel, the inset shows nuclear staining with DAPI in the same microscopic field as the main image.

Fig. 3.

Formoside induces receptor-mediated activation of CFTR via interaction with a GPCR. (A) Oocytes expressing the full-length clone and β₂AR, but not CFTR, did not respond to 5 μM formoside (F) or 1 μM isoproterenol (I) (n = 5). (Scale bar: 0.5 μA, 1 min.) (B) Current-voltage plot for isoproterenol-induced (red) and formoside-induced (blue) responses in oocytes expressing CFTR, β₂AR, and the full-length clone. (C and D) Multiple applications of 5 μM formoside to oocytes expressing CFTR, β₂AR, and clone A9-f4-230 (C) caused repeatable electrophysiological responses similar to the receptor-mediated responses to 1 μM isoproterenol (D) (n = 5) [Scale bars: C, 1.0 μA, 2 min; D, 0.5 μA, 2 min.) (E and F) Response to formoside requires a functional interaction between RL-TGR and a GPCR. Only cells expressing either β₂AR (E) (n = 5) or the rat aldehyde receptor OR-I7 (F) (n = 7) along with RL-TGR and CFTR responded robustly to formoside. Representative traces are provided in Fig. S3. Error bars represent SE. (G) Indirect immunofluorescence of heterologously expressed Strep II–RL-TGR with (Left) and without (Middle) β₂AR in HEK293 cells probed with anti–Strep II (green). Nuclei were stained with DAPI (blue). (Right) A confocal overlay image of Strep II–RL-TGR (green) in HEK293 cells. Individual image panels are shown in Fig. S4.

Fig. 4.

Proposed schematic of the coreceptor/GPCR complex. We hypothesize that RL-TGR, like known RAMPs, forms a complex with a GPCR to cooperatively bind ligand. The ligand-bound complex activates a signaling cascade through the GPCR's cognate G protein, resulting in the activation of signaling pathways that regulate ion channels, leading to the aversive behavior.