Figure 4
DOP-2 third intracellular loop (il 3 ) 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).