Osteocalcin binds to the GPRC6A Venus fly trap allosteric site to positively modulate GPRC6A signaling

GPRC6A is a member of the Family C G-protein coupled receptors that is activated by cations, L-amino-acids, osteocalcin (Ocn) peptide, and testosterone and functions as a master regulator of energy metabolism and sexual reproduction. Based on homology to related receptors, mGlur5 and CaSR, GPRC6A’s multiple ligand specificity is likely based on an orthosteric ligand binding domain in the bilobed venus fly trap (VFT) and two positive allosteric modulator (PAM) sites, one in the VFT and the other in the 7TM domain. In these studies, we show that Ocn acts as a PAM for GPRC6A, with binding to a site in the VFT distinct from the orthosteric site for calcium and amino-acids. In agreement with this model, alternatively spliced GPRC6A isoforms 2 and 3, which lack regions of the VFT, and mutations in the predicted Ocn binding site, K352E and H355P, prevented Ocn activation of GPRC6A. These observations provide a structural framework for understanding the ability of multiple distinct classes of compounds to activate GPRC6A and set the stage to develop novel small molecules to activate and inhibit this receptor. ### Competing Interest Statement The authors have declared no competing interest. * GPRC6A : G protein-coupled receptor family C group 6 member A Ocn : Osteocalcin VFT : Venus Flytrap CRD : Cysteine Rich Domain TM : Transmembrane cAMP : Cyclic adenosine monophosphate ERK : extracellular-signal-regulated kinase