Figure 4

Figure 4

The PBM motif in GIV-L binds to multiple PDZ proteins and enhances G-protein binding.A, schematic depicts the similarities between the sequences of the C-terminal PBMs (highlighted in red) of Daple and GIV-L and their respective immediate N-terminal flanking regions. While Daple's PBM is known to bind ParD3 and Dvl, whether GIV-L can bind is tested here. B, equal aliquots of lysates of HEK293T cells coexpressing various FLAG-tagged ParD3 constructs and GIV-L (wt) were subjected to immunoprecipitation assays using an anti-FLAG antibody. Bound proteins (left) and cell lysates (right) were assessed for ParD3 (FLAG) and GIV (myc) by immunoblotting (IB). C, equal aliquots of lysates of HEK293T cells coexpressing untagged Dvl1 and either myc-tagged GIV (wt) or GIV-L (wt or ΔPBM) were subjected to immunoprecipitation assays using an anti-myc antibody. Bound proteins (left) and cell lysates (right) were assessed for Dvl and GIV (myc) by immunoblotting (IB). D, equal aliquots of lysates of HEK293T cells coexpressing FLAG-tagged Gαi3 and either myc-tagged GIV (wt) or GIV-L (wt or ΔPBM) were subjected to immunoprecipitation assays using an anti-FLAG antibody. Bound proteins (top) and cell lysates (bottom) were assessed for Gαi3 (FLAG), GIV (myc), and endogenous Dvl by immunoblotting (IB). E, equal aliquots of lysates of HEK293T cells coexpressing untagged Dvl and either GST or GST-tagged GIV-L (CT) was incubated with glutathione agarose beads. Bound proteins (left) and cell lysates (right) were assessed for Gαi3, Dvl, or GST by immunoblotting (IB). F, schematic summarizing the differential impacts of binding of PDZ proteins to the C-terminal PBM motifs in Daple (left, middle) and GIV-L (right) on their ability to bind Gαi protein.