Project Summary: This MIRA proposal will combine work from our longstanding projects on the structure and function of adhesion G protein coupled receptors (or Family B2 GPCRs) and the molecular chaperones for G protein alpha subunits, Ric-8 proteins. Adhesion GPCRs are a large, 33-member family of receptors that transduce signals from cell to cell or cell to extracellular matrix adhesion events across the cell membrane to awaiting G proteins in order to elicit responses within the cell. Our work has advanced a mechanism for adhesion GPCR activation in which the extracellular adhesion domains become anchored to protein ligands and the cell that contains the GPCR or seven transmembrane domain (7TM) moves, by any number of means, in relation to this fixed anchor to dissociate the two halves of the adhesion GPCR. This dissociation event reveals a previously hidden tethered agonist of the GPCR/7TM domain that self-activates the receptor. This mode of activation seems common to most members of the adhesion GPCR family, but alternative modes of adhesion GPCR activation have also come to the fore of the field. Our goal in this proposal is to expand upon our mechanistic and structural work to decipher varied adhesion GPCR activation modes using biochemical, cell biological, and in vivo approaches, including work with two mouse models newly created for this proposal. We will focus on adhesion GPCRs that are present in cells that circulate in the blood, for example ADGRG1/GPR56 on platelets, as well as additional ADGRG subfamily members present on other circulating cell types. Circulating cells offer an advantage for interrogating adhesion GPCR action because the shear force component that dissociates the two fragments of the receptors, for the purpose of tethered-agonist-activation, is readily tractable. The sum of our work will be to distinguish tethered agonist -dependent and -independent modes of adhesion GPCR activation, which we argue is perhaps the most important contemporary question in the adhesion GPCR field. In parallel, we have assigned the function of Ric-8A and Ric-8B proteins as molecular chaperones that are required to collectively fold all G protein alpha subunits. This is a mature project for our lab, yet we have made recent breakthroughs in solving the structures of Ric-8/G protein guanine nucleotide-free complexes. We present preliminary evidence of a new Ric-8/G protein complex structure that will enable us to finally tackle a major outstanding question; What are the structural elements of mammalian Ric-8A and Ric-8B that define their specificities for different G protein subtypes? Combined with analysis of the single copy of an ancestral Ric-8 (Drosophila Ric-8) that seemingly crosses the lines and appears to fold all G protein subtypes, our work will provide new understanding of the substrate specificity rules, thereby providing a rationale for potentially targeting the Ric-8 chaperone system in contexts of G protein-driven disease.