The structural basis of allosteric signaling in G protein-coupled receptors (GPCRs) is important in guiding design of therapeutics and understanding phenotypic consequences of genetic variation. The Evolutionary Trace (ET) algorithm previously proved effective in redesigning receptors to mimic the ligand specificities of functionally distinct homologs. We now expand ET to consider mutual information, with validation in GPCR structure and dopamine D2 receptor (D2R) function. The new algorithm, called ET-MIp, identifies evolutionarily relevant patterns of amino acid covariations. The improved predictions of structural proximity and D2R mutagenesis demonstrate that ET-MIp predicts functional interactions between residue pairs, particularly potency and efficacy of activation by dopamine. Remarkably, although most of the residue pairs chosen for mutagenesis are neither in the binding pocket nor in contact with each other, many exhibited functional interactions, implying at-a-distance coupling. The functional interaction between the coupled pairs correlated best with the evolutionary coupling potential derived from dopamine receptor sequences rather than with broader sets of GPCR sequences. These data suggest that the allosteric communication responsible for dopamine responses is resolved by ET-MIp and best discerned within a short evolutionary distance. Most double mutants restored dopamine response to wild-type levels, also suggesting that tight regulation of the response to dopamine drove the coevolution and intramolecular communications between coupled residues. Our approach provides a general tool to identify evolutionary covariation patterns in small sets of close sequence homologs and to translate them into functional linkages between residues.allostery | G protein-coupled receptors | residue covariation | Evolutionary Trace I dentifying residues that coevolved to maintain or acquire fitness properties is critical for understanding protein structure, function, and evolution (1). Previous studies have shown that covarying residue pairs, those that exhibit correlated amino acid changes in large multiple sequence alignments, tend to form structural contacts (2-7), enhancing predictions of protein 3D structures (8-11). Covariation can also involve distal residues, but the function of these at-a-distance couplings is elusive and has been attributed to background noise, alternative protein conformations, or subunit interactions of protein homooligomers (5,7,12). Alternately, distal covarying residue pairs could indicate allosteric couplings (6,(13)(14)(15)(16)(17)(18).The possibility of capturing intramolecular allosteric communication by amino acid covariation analysis of protein family sequences has not been extensively explored. Nonproximal thermodynamic coupling between correlated residue pairs was noted in 274 PDZ domains (14), but the relationship to allostery is still debated (19,20). It may be that distinctive allosteric mechanisms, even among close homologs, limit the extraction of allosteric couplings from ...