Different mode of arrestin-3 binding at the human Y₁ and Y₂ 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 Y₁ and Y₂ receptor were identified. These receptors play an essential role in the regulation of feeding, energy homeostasis and cancer. The Y₁R displays high affinity to arr-3, which induces rapid internalization of the arrestin/receptor complex. In contrast, the Y₂R 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 Y₂R significantly, but not to the Y₁R suggesting different binding conformations. For the first time, the formation of a supercomplex consisting of Y receptor, Gα₀ protein and arrestin was studied by BRET-assay. We demonstrated that the Y₁R is able to bind Gα₀ protein as well as arr-3 simultaneously and internalizes as a supercomplex. For the Y₂R 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 Y₁R and Y₂R can be switched. Thus, we shed light on the specific spatio-temporal distribution of Gα₀ protein and arrestin in response to Y₁ versus Y₂ receptor activation and identified the molecular determinants.