Determining the activation of rho as an index of receptor coupling to G₁₂/₁₃ proteins

Heterotrimeric G proteins are composed of α, β, and γ subunits. G proteins can be activated by a large number of cell-surface hepathelical receptors and can transduce signals from these receptors to various intracellular signaling molecules. When G protein-coupled receptors are bound by their cognate ligand, interaction with specific subtypes of G protein leads to dissociation of the α subunit of the heterotrimeric G protein from the βγ dimer, and both Gα-GTP and Gβγ are capable of initiating their own signal transduction pathways. G proteins are functionally divided into four groups based on the nature of α subunit into G(s), G(i), G(q), and G₁₂ families. The members of the G₁₂ subfamily are G₁₂ and G₁₃. Increasing evidence indicates that G₁₂/₁₃ proteins play critical roles in various physiological functions. G₁₂ and G₁₃ regulate the small GTPase Rho through modulation of guanine nucleotide exchange factor (RhoGEF) activity to regulate various cellular responses, such as cytoskeletal changes and cell growth. Therefore, Rho activity can often represent a sensitive marker of G₁₂/₁₃ activity. Here, we describe the Rho activation assay to monitor the activity of G₁₂/₁₃ proteins.