Identification of G α 12 and G α 13 Determinants of Binding to Cell Adhesion Proteins

The trimeric G protein α‐subunits Gα12 and Gα13 have 66% amino acid homology but only partial overlap in their signaling function and downstream target proteins. Previously we reported that Gα12 and Gα13 bind the cytoplasmic domain of several cadherins, and other investigators have shown Gα13, but not Gα12, to bind the cytoplasmic tail of β3‐integrin. The switch I region of Gα13 has been implicated in binding to vascular endothelial (VE) cadherin and β3‐integrin, suggesting key residues arose in the G12/13 class to allow interaction with these cell‐surface adhesion proteins. To more precisely define determinants in the G12/13 class mediating these interactions, we utilized epitope‐tagged forms of Gα12 and Gα13 to examine their relative affinity for epithelial (E), neural (N), and VE cadherin, as well as β3‐integrin. Gα12 showed more robust interaction than Gα13 with both types of cell adhesion protein, and Gα12‐to‐Gα13 substitutions revealed several residues contributing to their different affinity for these proteins. Surprisingly, GFP tagging in the Gα12 helical domain disrupted binding to β3‐integrin but not cadherins, whereas Gα12 with a myc tag at the same location bound robustly to both proteins. These findings suggest that helical domain residues contributing to β3‐integrin binding are blocked by the larger GFP adduct but not the myc tag. Cassette substitutions in the Gα12 helical domain implicated several regions in binding cadherins, and these showed partial overlap with Gα12 mutations disruptive to β3‐integrin binding. These findings provide structural details of G12/13 engagement of cadherins and integrins and suggest distinct mechanisms of interaction with these adhesion proteins. Supported by the GSK Foundation and Lineberger Comprehensive Cancer Center