Different mode of arrestin-3 binding at the human Y and Y receptor.

GPCR internalization, which is induced by arrestin recruitment, is an important mechanism for the regulation of signaling and receptor quantity at the cell surface. In this study, differences in the mechanism of arrestin-3 (arr-3) recruitment to the neuropeptide Y and Y receptor were identified. These receptors play an essential role in the regulation of feeding, energy homeostasis and cancer. The YR displays high affinity to arr-3, which induces rapid internalization of the arrestin/receptor complex. In contrast, the YR has a lower affinity for arr-3. Internalization is induced by arrestin binding, but arr-3 is released from the receptor and remains at the membrane while the receptor internalizes. Moreover, the deletion of the finger loop region of arr-3 reduces its agonist-dependent recruitment to the YR significantly, but not to the YR suggesting different binding conformations. For the first time, the formation of a supercomplex consisting of Y receptor, Gα protein and arrestin was studied by BRET-assay. We demonstrated that the YR is able to bind Gα protein as well as arr-3 simultaneously and internalizes as a supercomplex. For the YR no supercomplex formation was observed. By substituting the C-terminus or specific residues within the intracellular loop 1 and 2 of the receptors, the arr-3 recruitment of the YR and YR can be switched. Thus, we shed light on the specific spatio-temporal distribution of Gα protein and arrestin in response to Y versus Y receptor activation and identified the molecular determinants.