CELLULAR HANDLING OF UNOCCUPIED AND AGONIST-STIMULATED CHOLECYSTOKININ RECEPTOR DETERMINED BY IMMUNOLOCALIZATION

Cellular handling of receptor molecules is an important mechanism for the regulation of appropriately sensitive hormone-stimulated signaling . Until now, our understanding of the cellular handling of the cholecy stokinin (CCK) receptor has been largely limited to following a tagged ligand through the cell. In the present work, we report the applicati on of unique CCK receptor antisera directed toward intracellular domai ns, which permitted the immunolocalization of this molecule independen tly of its occupation with ligand. The CCK receptor antisera were also useful in Western blotting and immunoprecipitation of this receptor. Unstimulated CCK receptors remained on the surface of both recombinant stable rat CCK-A receptor-bearing Chinese hamster ovary cell line (CH O-CCKR) cells and native rat pancreatic acinar cells and did not const itutively internalize. Agonist stimulation of the CHO-CCKR cells resul ted in the prompt internalization of a subset of surface receptors, re presenting those that were occupied with ligand. Unoccupied receptors remained on the surface, uninfluenced by the stimulated signaling path ways. Consistent with this, CCK receptor phosphorylation induced by 12 -O-tetradecanoylphorbol-13-acetate treatment did not stimulate recepto r internalization. After internalization, we observed substantial rece ptor recycling to the plasma membrane. These insights provide the firs t evidence that CCK receptor internalization occurs as a direct result of an induced conformational change and presumed bimolecular interact ion, rather than as an effect of a signaling event.