Dynein Light Chain Tctex-Type 1 Modulates Orexin Signaling through Its Interaction with Orexin 1 Receptor (original) (raw)
Figure 4
Regions of orexin receptors involved in their interaction with Dynlt1 and Dynlt3.
(A) Identification of a putative bipartite Dynlt1-binding motif in orexin receptors. The proximal portion of the structure shown here, located in the third intracellular loop of orexin receptors, is not included in the soluble orexin receptors CTD containing the distal part and used for yeast two-hybrid assays (Fig. 1 and 3). Amino acid numbering refers to mouse sequences. Amino acids of the consensus delineated from other Dynlt1-binding proteins are shown in bold, [30], [31]. In the OX1R CTD mutant, two conserved Thr were mutated to Ala (409 and 412, numbering from full-length OX1R). (B, C, D) β-galactosidase (top panels) and HIS3 (bottom panels) assays were performed on yeast transformed with plasmids expressing different combinations of orexin receptor and Dynlt1/Dynlt3. Interactions of OX1R CTD with Dynlt1 and Dynlt3 are reduced when Thr 409 and 412 of OX1R CTD are mutated into Ala. Deleting the extra amino acids of OX2R CTD favors its interaction with Dynlt1, while deleting the next 10 amino acids (comprising the distal part of the Dynlt1-binding motif) abolishes this effect. (E) Summary of OX1R CTD and OX2R CTD constructs tested and their relative interaction strength with Dynlt1 and Dynlt3. OX1R CTD T409, 412A, OX1R CTD with T409 and T412 mutated into alanine residues; OX1R CTD Δ407–416, OX1R CTD lacking the last 10 a.a.; OX1R CTD Δ397–416, OX1R CTD lacking the last 20 a.a.; OX1R CTD Δ387–416, OX1R CTD lacking the last 30 a.a.; OX2R CTD Δ433–460, OX2R CTD lacking the extra 28 a.a. compared to OX1R CTD; OX2R CTD Δ423–460, OX2R CTD lacking the last 38 a.a.; OX2R CTD Δ413–460, OX2R CTD lacking the last 48 a.a.; OX2R CTD Δ403–460, OX2R CTD lacking the last 58 a.a. ND, not determined. Experiments were performed 3 times and the average is presented. **: p<0.01 vs transformation with wild-type OX1R CTD or OX2R CTD.