Molecular determinants underlying differential recruitment of p115RhoGEF and PDZRhoGEF to activated Gα ₁₃

Heterotrimeric G proteins, particularly Gα ₁₂ and Gα ₁₃ , are pivotal regulators of cellular signaling pathways. Their direct downstream effectors, which include p115RhoGEF and PDZRhoGEF, engage downstream signaling via Rho activation. Yet the molecular determinants that dictate their differential recruitment by Gα _12/13 are not fully understood. Here, we combined quantitative computational residue-level analysis with site-directed mutagenesis and bioluminescence resonance energy transfer (BRET)-based assays to dissect Gα ₁₃ interactions with these RhoGEFs. We mapped the contributions of individual residues to binding and identified specific Gα ₁₃ residues in its helical domain, switch regions, and effector-binding site as key yet differential contributors to p115RhoGEF and PDZRhoGEF recruitment. Experimental validation with BRET confirmed that changes in many Gα ₁₃ residues impact p115RhoGEF more substantially than PDZRhoGEF, underscoring the specificity of Gα ₁₃ interactions with p115RhoGEF. Investigation of the p115RhoGEFs identified critical residues that contribute to interactions with Gα ₁₃ and Gα ₁₂ . Our findings highlight residue-level differences in the molecular interactions of Gα ₁₃ with p115RhoGEF and PDZRhoGEF, providing insights into the specificity and regulation of Gα ₁₃ -mediated signaling pathways. The resulting residue-level maps lay the groundwork for development of selective therapeutic strategies targeting Gα ₁₃ -RhoGEF interactions.