DUAL PATHWAYS OF INTERNALIZATION OF THE CHOLECYSTOKININ RECEPTOR

Receptor molecules play a major role in the desensitization of agonist -stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, a nd internalization, yet the cellular compartments in which these event s occur and their interrelationships are unclear. In this work, we foc us on the cholecystokinin (CCK) receptor, which has been well characte rized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe recep tor localization in a CCK receptor-bearing CHO cell line. In the unsti mulated state, receptors were diffusely distributed over the plasmalem ma. Agonist occupation stimulated endocytosis via both clathrin-depend ent and independent pathways. The former was predominant, leading to e ndosomal and lysosomal compartments, as well as recycling to the plasm alemma. The clathrin-independent processes led to a smooth vesicular c ompartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion large ly eliminated clathrin-dependent endocytosis, while not interfering wi th agonist-stimulated receptor movement into subplasmalemmal smooth ve sicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphoryl ation, which we have previously described (Klueppelberg, U. G., L. K. Gates, F. S. Gorelick, and L. J. Miller. 1991. J. Biol. Chem. 266:2403 -2408; Lutz, M. P., D. I. Pinon, L. K. Gates, S. Shenolikar, and L. J. Miller. 1993. J. Biol. Chem. 268:12136-12142). The rapid onset and pe ak of receptor phosphorylation after agonist occupation correlates bes t with a plasmalemmal localization, while stimulated receptor phosphat ase activity correlates best with receptor residence in intracellular compartments. We postulate that the smooth vesicular compartment adjac ent to the plasmalemma functions for the rapid resensitization of the receptor, while the classical clathrin-mediated endocytotic pathway is key for receptor downregulation via lysosomal degradation, as well as less rapid resensitization.