Dynein Light Chain Tctex-Type 1 Modulates Orexin Signaling through Its Interaction with Orexin 1 Receptor (original) (raw)
Figure 5
The effect of Dynlt1 mutations and OX1R mutations on their interaction in mammalian cells.
(A) HEK293 cells were transfected with the indicated constructs and stimulated with orexin-A (OX-A) or vehicle (water) for 30 min. Whole-cell lysates were then subjected to IP as described in Fig. 2. OX1R interaction with Dynlt1 is not disrupted by the presence of deletion mutants of Dynlt1 after addition of vehicle, but it is reduced (compared to wild-type Dynlt1) after stimulation with OX-A. (B) HEK293 cells were transfected with the indicated constructs and whole-cell lysates were subjected to IP. Removing the CTD of OX1R abolishes the interaction of OX1R with Dynlt1. (C, D) Co-IP experiment performed on whole-cell lysates obtained from HEK293 cell transfected with the indicated constructs (Myc-Dynlt1 and V5-OX1R deletion mutants). Removing the last 10 amino acids from OX1R abolishes its interaction with Dynlt1, while mutating two threonine residues for alanine in the putative Dynlt1-binding motif (Fig. 4A) blunts the amount of Myc-Dynlt1 co-immunoprecipitated with V5-OX1R. (E) Quantification of Western blotting bands obtained in panels B, C and D. All transfection conditions and co-IP experiments were performed at least twice.