The main process of downregulation of G protein-coupled receptors is desensitization by which the receptor is extruded from the plasma membrane and directed to the endosomal compartment for recycling. Typically, the first step of this phenomenon consists in the recruitment of the protein β-arrestin induced by the agonist. Melatonin receptors undergo the same process: melatonin leads to the recruitment of β-arrestin and is subsequently sent away from the membrane, leading to a de facto stop of the melatonin receptor-mediated G protein signaling, because the receptors are not at the membrane level to receive the message brought by melatonin. The way one can measure this recruitment is based on the elegant technique of enzyme fragment complementation by which two parts of an enzyme are fused to two partners and reform an active enzyme upon the formation of the complex between these two partners. The basic way to set up this technique is presented here.

The main step of classical desensitization of a receptor, by mean of its disappearance from the plasma membrane, is its internalization. This is a key factor in the regulation of agonist-mediated signaling pathways, as it most of the time stops the activation of the receptor. Internalization is thus important to evaluate, as a complementary information for a natural ligand or an alternative synthetic agonist. Enzyme fragment complementation is an elegant but delicate way to measure this phenomenon, by fusing two complementary parts of an enzyme to two partners, and to measure the activity of the reconstituted enzyme upon complexation of the partners. In the present chapter, using two parts of β-galactosidase, one fused to the C-terminus of the MT1 receptor, the other to an endosomal protein, one can measure the formation of the complex; thus, the transfer of the receptor to the endosome from which MT1 will be recirculated.