Despite recent advances in structural definition of GPCR–G protein complexes, the basis of receptor selectivity between G proteins remains unclear. The Gα
12
and Gα
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 Gα
13
but weakly to Gα
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 Gα
13
. Incorporating Gα
12
/Gα
13
chimeras and individual residue swap mutations into these sensors defined that selectivity between Gα
13
and Gα
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 Gα
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 Gα
q
, resulting in the gain of coupling to GPR35. These studies demonstrate that Gα
13
is markedly the most effective G protein for interaction with GPR35 and that selection between Gα
13
and Gα
12
is dictated largely by a single conservative amino acid variation.—Mackenzie, A. E., Quon, T., Lin, L.-C., Hauser, A. S., Jenkins, L., Inoue, A., Tobin, A. B., Gloriam, D. E., Hudson, B. D., Milligan, G. Receptor selectivity between the G proteins Gα
12
and Gα
13
is defined by a single leucine-to-isoleucine variation.
The present study demonstrates the usefulness of ITS2 PCR-RFLP coupled with pre-selected restriction enzymes for practical and accurate authentication of S. flavescens Ait. The technique is also suitable for analysing S. flavescens Ait. mixed with other adulterants.
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