Figure 1
Constructs used for split ubiquitin assays. Bait protein fusion was made with the C-terminal part of ubiquitin (Cub) followed by artificial transcription factor LexA-VP16 at the C-terminus of DOP-2XL (1, 2). Prey protein fusions were constructed with the mutant N-terminal part of ubiquitin (NubG) either at the C terminus of GPA-14 (3) or at the N-terminus (4). If bait and prey interact NubG and Cub are forced into close proximity, resulting in the reconstitution of split-ubiquitin and release of LexA-VP16 transcriptional factor that leads to transcriptional readout, resulting in growth of yeast on selective medium and color development in a β-galactosidase assay.
Figure 2
Interaction studies of dopamine receptor DOP-2XL with Gα protein GPA-14. (A) Split-ubiquitin based pair-wise interaction. Yeast two-hybrid screen with full-length DOP-2XL yielded GPA-14 as a positive interaction partner that was validated by pair-wise interaction between dop-2c (cloned in pBT3-STE) and gpa-14 (cloned in pPR3-N and pPR3-STE). The interaction was determined by growth assay on selective growth media and X-gal filter overlay assay. For the growth assay, selected yeast colonies were streaked onto a master plates (plate 1, SD/-Leu-Trp), and selective plates (plate 2, SD/-Ade-His-Leu-Trp) containing 1 mM 3-AT. Absence of adenine from the selection medium is useful since in the absence of a protein-protein interaction, the ADE2 reporter gene is not transcribed and therefore, a red-colored intermediate accumulates in the adenine metabolic pathway and gives red color to the colony (plate 1). To perform X-gal filter lift-off assay (plate 3, X-gal) colonies from master plate were replica transferred to 3 mM filter paper and assay was performed as described by Dualsystems. The growth results and X-gal assay results are interpreted as good interaction (+), weak interaction (+/-), or no interaction (-). (B) In-vitro interaction between DOP-2XL and GPA-14. GST-tagged GPA-14 and GPA-15 (~ 72 KDa) was purified by immobilization with paramagnetic GST particles (data not shown). DOP-XL protein (~94 KDa) expression was obtained by in-vitro transcription/translation method (lane1). DOP-2 XL was incubated either with purified GST:GPA-14 or with purified GST:GPA-15, GST, paramagnetic beads, as controls. At the end of the incubation the beads were collected from the mixtures and washed. The eluted samples were applied to SDS PAGE and transferred to PVDF membrane and probed colorimetrically. In-vitro synthesized DOP-2XL was pulled down with Magne-GST beads when incubated with GST:GPA-14 (lane 2) but not when incubated with GST alone (lane 3), or with magnetic beads only (lane 4); however, a weak band is visible with GST:GPA-15 (lane 5) which is likely due to non-specific interaction.
Figure 3
Domain analysis of human DRD2Longer and C. elegans DOP-2XL dopamine receptors. (A) Schematic representation of receptor domains. DRD2Longer and CeDOP-2XL intracellular loop (il) are represented as blue shaded boxes and Nt and Ct are shown as green and red boxes, respectively. The numbers indicate the position of each intracellular loop in both the receptors. DOP-2XL il3 (224-766 aa) consists of 546 amino acid residues that is more than 3 times the length of DRD2 il3 (214-374 aa) with 160 amino-acid length. il1and il2 were almost comparable in size with less than 10 amino-acid difference. (B) Sequence comparison of receptor domains. Sequence alignment of DOP-2XL (WormBase accession no WP:CE45633) and DRD2Longer (UniProt accession no P14416-3) was performed based on their predicted GPCR structures. The domains used for alignment were il2 and il3 (il3N) & (il3C) regions found to be necessary for Gi coupling.
Figure 4
DOP-2 third intracellular loop (il3) interacts with GPA-14. (A) Schematic representation of DOP-2XL truncated protein constructs. DOP-2XL truncated region in each construct is indicated by amino-acid residue positions. Panel 1-4 are constructs, DOP-2IL-CI- DOP-2IL-CIV for yeast two-hybrid interactions. DOP-2IL-CI (amino acids 64-849) consisted of all the domains but N-terminal domain and transmembrane 1 was absent. DOP-2IL-CII protein (124-849 amino acids) starts at the junction of predicted transmembrane 2 and il2. DOP-2IL-CIII (amino acids 224-849), truncated protein was lacking domains il1 and il2. DOP-2IL-CIV truncated protein (821-849 amino acids) is just the C-terminal intracellular region beyond transmembrane 7 without any loop. Panels 5 & 6 represent DOP-2-CV and DOP-2-CVI used for His pull down assay. DOP-2-CV (amino acids1-182) expressed il1+il2 and DOP-2-CVI (amino acids 183-849) consisted of il3+Ct region (B) Interaction between truncated DOP-2 bait constructs and GPA-14 prey proteins. dop-2c FL was cloned in pBT3-STE and truncated domain constructs were made in pBT3-SUC vector to be used as bait, gpa-14 was cloned in two different prey vectors pPR3-N (upper panel) and pPR3-STE (lower panel). Interaction was determined by growth assay on selective growth media, plate 1 (SD/-Leu-Trp), plate 2 (SD/-Ade-His-Leu-Trp), X-Gal filter overlay assay, plate 3 (X-gal) and demonstrated as (+) good interaction; weak interaction (+/-); no interaction (-). Sector-I represents positive interaction control, sector-VIII was negative-control in both the cases, and sectors II, III, IV, V and VI were test interactions. (C). Interaction between DOP-2 truncated constructs and Gα-proteins by His pull-down assay. Lane 1 shows in-vitro translated Gα-His fusion proteins (GPA-14, GPA-15 and GSA-1) and DOP-2XL constructs (CV, CVI). DOP-2XL constructs were incubated with His tagged GPA-14 or with control proteins His:GPA-15/His:GSA-1 at 4°C. After allowing these proteins to interact, nickel resin (Mag Z particles, Promega) was added. At the end of this incubation, beads were collected by using magnetic stand and washed and eluted, and the sample was resolved by SDS-PAGE. Colorimetric detection of interaction complex showed that DOP-2-CVI (lane 3) but not DOP-2-CV (lane 2) was pulled down with GPA-14-His protein. Both DOP-2-CV & DOP-2-CVI showed no binding with control Gα proteins GPA-15 and GSA-1 (lanes 4, 5, 6 & 7).