AJP - Gastrointestinal and Liver Physiology, Vol 272, Issue 3 488-G497, Copyright © 1997 by American Physiological Society

ARTICLES

Cellular handling of unoccupied and agonist-stimulated cholecystokinin receptor determined by immunolocalization

C. Fischer de Toledo, B. F. Roettger, C. Morys-Wortmann, W. E. Schmidt and L. J. Miller
Center for Basic Research in Digestive Diseases, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.

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.

This article has been cited by other articles:

				J. R. Reeve Jr., S. V. Wu, D. A. Keire, K. Faull, P. Chew, T. E. Solomon, G. M. Green, and T. Coskun Differential bile-pancreatic secretory effects of CCK-58 and CCK-8 Am J Physiol Gastrointest Liver Physiol, March 1, 2004; 286(3): G395 - G402. [Abstract] [Full Text] [PDF]

				D. M. Shin, X. Luo, T. M. Wilkie, L. J. Miller, A. B. Peck, M. G. Humphreys-Beher, and S. Muallem Polarized Expression of G Protein-coupled Receptors and an All-or-None Discharge of Ca2+ Pools at Initiation Sites of [Ca2+]i Waves in Polarized Exocrine Cells J. Biol. Chem., November 16, 2001; 276(47): 44146 - 44156. [Abstract] [Full Text] [PDF]

				X.-Q. Ding, R. V. Rao, S. M. Kuntz, E. L. Holicky, and L. J. Miller Impaired Resensitization and Recycling of the Cholecystokinin Receptor by Co-expression of its Second Intracellular Loop Mol. Pharmacol., April 13, 2001; 58(6): 1424 - 1433. [Abstract] [Full Text]

				R. V. Rao, E. L. Holicky, S. M. Kuntz, and L. J. Miller CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking Am J Physiol Cell Physiol, December 1, 2000; 279(6): C1986 - C1992. [Abstract] [Full Text] [PDF]

				B. F. Roettger, D. I. Pinon, T. P. Burghardt, and L. J. Miller Regulation of lateral mobility and cellular trafficking of the CCK receptor by a partial agonist Am J Physiol Cell Physiol, March 1, 1999; 276(3): C539 - C547. [Abstract] [Full Text] [PDF]

				W. Y. Go, E. L. Holicky, E. M. Hadac, R. V. Rao, and L. J. Miller Identification of a domain in the carboxy terminus of CCK receptor that affects its intracellular trafficking Am J Physiol Gastrointest Liver Physiol, July 1, 1998; 275(1): G56 - G62. [Abstract] [Full Text] [PDF]