Receptor selectivity between the G proteins Gα12 and Gα13 is defined by a single leucine-to-isoleucine variation

Despite recent advances in structural definition of GPCR-G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The G alpha(12) and G alpha(13) subtypes together form the least studied group of heterotrimeric G proteins. G protein-coupled receptor 35 (GPR35) has been suggested to couple efficiently to G alpha(13) but weakly to G alpha(12). Using combinations of cells genome-edited to not express G proteins and bioluminescence resonance energy transfer-based sensors, we confirmed marked selectivity of GPR35 for G alpha(13). Incorporating G alpha(12)/G alpha(13) chimeras and individual residue swap mutations into these sensors defined that selectivity between G alpha(13) and G alpha(12) was imbued largely by a single leucine-to-isoleucine variation at position G.H5.23. Indeed, leucine could not be substituted by other amino acids in G alpha(13) without almost complete loss of GPR35 coupling. The critical importance of leucine at G.H5.23 for GPR35-G protein interaction was further demonstrated by introduction of this leucine into G alpha(q), resulting in the gain of coupling to GPR35. These studies demonstrate that G alpha(13) is markedly the most effective G protein for interaction with GPR35 and that selection between G alpha(13) and G alpha(12) is dictated largely by a single conservative amino acid variation.