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 cholecystokinin (CCK) receptor has been largely limited to following a tagged ligand through the cell. In the present work, we report the application of unique CCK receptor antisera directed toward intracellular domains, which permitted the immunolocalization of this molecule independently 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 (CHO-CCKR) cells and native rat pancreatic acinar cells and did not constitutively internalize. Agonist stimulation of the CHO-CCKR cells resulted in the prompt internalization of a subset of surface receptors, representing those that were occupied with ligand. Unoccupied receptors remained on the surface, uninfluenced by the stimulated signaling pathways. Consistent with this, CCK receptor phosphorylation induced by 12-O-tetradecanoylphorbol-13-acetate treatment did not stimulate receptor internalization. After internalization, we observed substantial receptor recycling to the plasma membrane. These insights provide the first evidence that CCK receptor internalization occurs as a direct result of an induced conformational change and presumed bimolecular interaction, rather than as an effect of a signaling event.
Fischer de Toledo
C
C
Center for Basic Research in Digestive Diseases, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Roettger
B F
BF
Morys-Wortmann
C
C
Schmidt
W E
WE
Miller
L J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D007158:Immunologic Techniques; D008297:Male; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism* / Q000648:ultrastructure; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D011949:Receptors, Cholecystokinin / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D011949:Receptors, Cholecystokinin; D013755:Tetradecanoylphorbol Acetate