Figure 5. Direct protein-protein interaction between the third intracellular loop of M3R and the DEP domain of RGS7.

(A) The M3 receptor deletion mutant lacking the 3rd intracellular loop (M3R-short) was transfected into CHO-K1 cells together with Lac Z or Gβ₅-RGS7. Changes in Ca²⁺ were recorded upon the application of 100nM Cch (black bar). The grey line represents a Ca²⁺ transient recorded from cells transfected with M3R-short alone. Black line represents the M3R-short transfected together with Gβ₅-RGS7. (B) GST fusions of the i3 loops of the M1, M2, M3 and M5 receptors or GST were immobilized on glutathione agarose beads. The beads were incubated with the extract from CHO-K1 cells transfected with YFP DEP, as described in Materials and Methods. After the slurry was spun down and unbound material was collected, the resin was washed and eluted with SDS sample buffer. The unbound (U) and eluted (E) material was analyzed by western blot using the anti-GFP antibody. (C) Schematic representation of the sequence of the entire M3R i3 loop. The approximate location of the binding sites for Gαq, Gβγ, SET, regions phosphorylated by GRK2 and casein kinase α (CKα), calmodulin (CaM), and β-arrestin (β-Arr) are indicated according to the literature (see text). The truncated GST-fused M3R i3 loop regions as are shown under the full-length M3R i3 loop. Filled boxes designate constructs that bound the RGS7 DEP domain and the open box shows the construct that did not bind to the DEP domain. The representative results of the pull-down assays obtained with these GST-fusion proteins are shown to the right.