The adhesion G protein-coupled receptors (ADGRs/class B2 G protein-coupled receptors) constitute an ancient family of G protein-coupled receptors that have recently been demonstrated to play important roles in cellular and developmental processes. Here, we describe a first insight into the structure-function relationship of ADGRs using the family member ADGR subfamily G member 4 (ADGRG4)/GPR112 as a model receptor. In a bioinformatics approach, we compared conserved, functional elements of the well-characterized class A and class B1 secretin-like G protein-coupled receptors with the ADGRs. We identified several potential equivalent motifs and subjected those to mutational analysis. The importance of the mutated residues was evaluated by examining their effect on the high constitutive activity of the N-terminally truncated ADGRG4/GPR112 in a 1-receptor-1-G protein Saccharomyces cerevisiae screening system and was further confirmed in a transfected mammalian human embryonic kidney 293 cell line. We evaluated the results in light of the crystal structures of the class A adenosine A2A receptor and the class B1 corticotropin-releasing factor receptor 1. ADGRG4 proved to have functionally important motifs resembling class A, class B, and combined elements, but also a unique highly conserved ADGR motif (H3.33). Given the high conservation of these motifs and residues across the adhesion G protein-coupled receptor family, it can be assumed that these are general elements of adhesion GPCR function.-Peeters, M. C., Mos, I., Lenselink, E. B., Lucchesi, M., IJzerman, A. P., Schwartz, T. W. Getting from A to B-Exploring the activation motifs of the class B adhesion G protein-coupled receptor subfamily G member 4/GPR112.