The N-terminal domain of GPR61, an orphan G-protein-coupled receptor, is essential for its constitutive activity

G-protein-coupled receptor 61 (GPR61) is an orphan receptor that is abundantly expressed in the brain, which suggests its involvement in various physiological functions in the central nervous system. It couples with Gs and shows constitutive activity. To investigate the role of the N-terminal segment in the constitutive activity of GPR61, we measured [(35)S]GTPgammaS binding using a GPR61-Gs fusion protein and derivatives that had a deletion or alanine mutation in the N-terminal segment. We found that deletion of the N-terminal 25 amino acids and the V19A mutation in GPR61 impaired its constitutive activity. Moreover, the loss of the constitutive activity of the mutants could be restored by adding a fusion protein containing a C-terminal CD8 single transmembrane domain and the N-terminal 48-amino-acid segment of GPR61, i.e., CD8-48. We conclude that the N-terminal domain of GPR61 is required for maintaining its constitutive activity and functions as a tethered intramolecular ligand.