Figure 1. G protein β subunits bind to DDB1 and CUL4 independent of Gγ proteins.

(A) G protein β subunits contain the DDB1-binding WD40 (DWD) motif. The amino acid sequences spanning the DWD box from five human Gβ proteins are aligned (Gβ1, NCBI number: NP_002065.1; Gβ2: NP_005264.2; Gβ3: NP_002066.1; Gβ4: NP_067642.1; Gβ5: NP_006569.1). Also included are three well-characterized human DWD proteins, DDB2 (NP_000098.1), CSA (NP_000073.1), and CDT2 (NP_057532.3). Highly conserved residues are in bold, and residues essential for DDB1 binding, Arg273 in DDB2 and Arg214 in Gβ2, are circled.

(B) Gβ proteins bind with DDB1-CUL4A. 293T cells were co-transfected with plasmid expressing indicated proteins. Protein-protein bindings were determined by co-immunoprecipitation (co-IP). (‘α-Flag’ means anti-Flag antibody, the same below).

(C) The N-terminal domain of CUL4A is required for binding with Gβ2. 293T cells were co-transfected with plasmid expressing indicated proteins and protein-protein bindings were determined by co-IP (‘5% input’ means 5% total protein for IP experiments were loaded, the same below).

(D) The conserved Arg214 in the DWD box of Gβ2 is required for the binding with CUL4A. 293T cells were co-transfected with plasmids expressing indicated proteins and protein-protein bindings were determined by co-IP assay.

(E, F) Endogenous Gβ2 binds with CUL4A in HEK293 cells (E) and primary rat cardiomyocytes (F) as determined by the co-IP assay.

(G) Gβ2 binds with CUL4A independent of Gγ. 293T cells were co-transfected with plasmids expressing indicated proteins and protein-protein bindings were determined by co-IP.