Oligomerisation of C. elegans olfactory receptors, ODR-10 and STR-112, in yeast

Abstract

It is widely accepted that vertebrate G-Protein Coupled Receptors (GPCRs) associate with each other as homo- or hetero-dimers or higher-order oligomers. The C. elegans genome encodes hundreds of olfactory GPCRs, which may be expressed in fewer than a dozen chemosensory neurons, suggesting an opportunity for oligomerisation. Here we show, using three independent lines of evidence: co-immunoprecipitation, bioluminescence resonance energy transfer and a yeast two-hybrid assay that nematode olfactory receptors (ORs) oligomerise when heterologously expressed in yeast. Specifically, the nematode receptor ODR-10 is able to homo-oligomerise and can also form heteromers with the related nematode receptor STR-112. ODR-10 also oligomerised with the rat I7 OR but did not oligomerise with the human somatostatin receptor 5, a neuropeptide receptor. In this study, the question of functional relevance was not addressed and remains to be investigated.

Figure 1. Yeast cells expressing ODR-10-GFP².

Arrows indicate the plasma membrane and cytoplasmic localisation. Bar: 11.9 µm. Image was obtained with a Leica SP2 confocal laser scanning microscope using excitation at 488 nm.

Figure 2. Three lines of evidence for formation of ODR-10 homo-oligomers.

A. Co-immunoprecipitation of a pair of tagged chemoreceptor subunits. B. ODR-10-Rluc co-expressed with SSTR5-GFP² as a negative control. Following membrane extraction with digitonin and immunoprecipitation with an anti-GFP² antibody, as described in Materials and Methods, luciferase activity was measured using 5 µM Coelenterazine H with an emission filter of 475±30 nm. Values represent means ± SD of experiments performed on three independent transformants (**denotes significance at P≤0.001), “b” is significantly different from “a”. C. BRET² ratios measured to test for homo-dimerisation of ODR-10 in intact yeast cells. Values represent means ± SD (n = 3) (**denotes significance at P≤0.001), “b” is significantly different from “a”. Indicated constructs in both A and C were expressed separately, or mixed (+), or co-expressed (/). D. ODR-10 homo-oligomerises in the split-ubiquitin yeast two-hybrid system. The C-terminal half of ubiquitin (Cub) was fused to the C-terminus of the full-length cDNA of odr-10 (ODR-10::Cub). The N-terminal half of ubiquitin (NubG) was fused to the C- termini of full-length cDNA of odr-10 (ODR-10::NubG). The interaction of ODR-10::Cub with pOST1:NubI served as a positive control to ensure the correct topology of the fusion protein. The interaction of ODR-10::Cub with the empty vector (pPR3-STE) served as a negative control. Yeast transformants containing both a Cub fusion and a NubG fusion construct were grown on drop out media (SD -Leu and -Trp) (Figure S2A) and selective medium lacking histidine (SD -Leu, -Trp and -His) containing 35 mM 3-Amino-1, 2, 4-triazole (3-AT). β-galactosidase assays were performed to verify interactions. Cells were spotted as one-tenth dilutions starting at Abs_600nm = 1. E. ODR-10 does not hetero-oligomerise with SSTR5 in the split-ubiquitin yeast two-hybrid system. Growth and β-galactosidase activity of yeast cells expressing ODR-10:Cub and SSTR5:NubG fusions. The interaction of SSTR5::NubG with the empty vector (pBT3-STE) served as a negative control. Yeast transformants containing both a Cub fusion and a NubG fusion constructs were grown on drop out media (SD -Leu and -Trp) (Figure S2E) and selective medium lacking histidine (SD -Leu, -Trp and -His) containing 35 mM 3-Amino-1, 2, 4-triazole (3-AT). β-Galactosidase assays were performed to verify interactions. Cells were spotted as one-tenth dilutions starting at Abs_600nm = 1.

Figure 3. Constructs used for split-ubiquitin assays.

A. Bait fusion proteins were made with the C-terminus of ubiquitin (Cub) linked to the synthetic transcription factor LexA-VP16 at the C-termini of ODR-10, STR-112 and I7 in the pBT3-STE plasmid. The STE2 sequence improves translation of protein constructs. B. Prey protein fusions were constructed with the mutant N-terminal part of ubiquitin (NubG) at the C termini of the SSTR5, STR-112, ODR-10 and I7. If bait and prey interact, NubG and Cub are brought into close proximity, resulting in the reconstitution of functional ubiquitin and release of the LexA-VP16 transcriptional factor, which leads to transcriptional readout, resulting in growth of host yeast cells on the selective medium (SD-L-T-H) or color development in a β-galactosidase assay.

Figure 4. ODR-10 hetero-oligomerises with STR-112 in the split-ubiquitin yeast two-hybrid system.

Growth and β-Galactosidase activity of yeast cells expressing various combinations of Cub and NubG fusions. The control plasmids were pPR3-STE and pBT3-STE. Yeast transformants containing both Cub fusion and NubG fusion constructs were grown on drop out media (SD -Leu and -Trp) (Figure S2B) and selective medium lacking histidine (SD -Leu, -Trp and -His) containing 35 mM 3-Amino-1, 2, 4-triazole (3-AT). β-galactosidase assays were performed to verify interactions. Cells were spotted as one-tenth dilutions starting at Abs_600nm = 1.

Figure 5. ODR-10 hetero-oligomerises with I7.

A. Co-immunoprecipitation of pairs of tagged chemoreceptor subunits. Values represent means ± SD of experiments performed in triplicate with three independent transformants for each condition (**denotes significance at P≤0.001), “b” is significantly different from “a”. B. BRET² ratios measured to test for hetero-dimerisation of ODR-10 and I7 in intact yeast cells. Values represent means ± SD (n = 3) (**denotes significance at P≤0.002), “b” is significantly different from “a”. Indicated constructs in both A and B were expressed separately, or mixed (+), or co-expressed (/). C. ODR-10 hetero-oligomerises with I7 in the split-ubiquitin yeast two-hybrid system. Growth and β-galactosidase activity of yeast cells expressing ODR-10:Cub and I7:NubG fusions. Yeast transformants containing both Cub fusion and NubG fusion constructs were grown on drop out media (SD -Leu and -Trp) (Figure S2C) and selective medium lacking histidine (SD -Leu, -Trp and -His) containing 35 mM 3-Amino-1, 2, 4-triazole (3-AT). β-galactosidase assays were performed to verify interactions. Cells were spotted as one-tenth dilutions starting at Abs_600nm = 1.

Figure 6. I7 forms homo-oligomers.

A. Co-immunoprecipitation of a pair of tagged chemoreceptor subunits. Values represent means ± SD of experiments performed in triplicate with three independent transformants for each condition (**denotes significance at P≤0.001), “b” is significantly different from “a”. B. BRET² ratios measured to test for homo-oligomerisation of I7 in intact yeast cells. Values represent means ± SD of experiments performed in triplicate with three independent transformants for each condition. (*denotes significance at P≤0.01). Indicated constructs in both A and B were expressed separately, or mixed (+), or co-expressed (/). C. I7 homo-oligomerises in the split-ubiquitin yeast two-hybrid system. Growth and β-galactosidase activity of yeast cells expressing I7:Cub and I7:NubG fusions. The interaction of I7::Cub with the empty vector (pPR3-STE) served as a negative control. Yeast transformants containing both a Cub fusion and a NubG fusion constructs were grown on drop out media (SD -Leu and -Trp) (Figure S2D) and selective medium lacking histidine (SD -Leu, -Trp and -His) containing 35 mM 3-Amino-1,2,4-triazole (3-AT). β-galactosidase assays were performed to verify interactions. Cells were spotted as one-tenth dilutions starting at Abs_600nm = 1.