The class C G protein-coupled receptor GPRC6A is a putative nutrient-sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization, whereas the agonist-induced effects were imperceptible. Moreover, postendocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient-sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure.The G protein-coupled receptor, class C, group 6, member A (GPRC6A), 2 is a nutrient-sensing G protein-coupled receptor (GPCR) belonging to class C. It is activated by basic L-ā£-amino acids and divalent cations, which trigger coupling to the G q protein and thereby an increase in the intracellular levels of the second messengers inositol trisphosphate and Ca 2Ļ© (1-8). Other ligands and signaling pathways have been reported (3, 9 -13); however Rueda et al. (8) and we (7) have not been able to confirm these findings. The GPRC6A receptor displays a broad but low expression profile in human, mouse, and rat (2,5,10,11,14,15), thus giving little indication to the physiological role of the receptor. Several groups have conducted studies using GPRC6A knock-out mice to elucidate the physiological function of the receptor, and although results differ between knockout mouse models, they altogether suggest an involvement in metabolism and endocrine regulation (6, 10, 11, 16 -18).Over the years, it has become evident that GPCR signaling is much more complex than once believed. For most receptors, a variety of ligands and intracellular signaling pathways are available, and numerous regulatory mechanisms are thus required for obtaining specific biological responses (19).