Cellular handling of the unoccupied and agonist-stimulated
cholecystokinin receptor determined by immunolocalization.
Fischer, Carlos, De Toledo, Belinda F. Roettger, Corinna Morys
-Wortmann, Wolfgang E. Schmidt, Laurence J. Miller.
Center for Basic Research in Digestive Diseases, Mayo Clinic and
Mayo Foundation, Rochester, Minnesota 55905, Max-Planck-Institut
F[umlaut]ur Experimentelle Medizin, Abteilung Immunchemie, D-37075
G[diaeresis]ottingen, Hermann-Rein-Strasse 3, Germany and Labor fur
Molekulare Gastroenterologie, I. Medizinische Klinik der CAU Kiel, D
-24105 Kiel, Germany
APStracts 3:0184G, 1996.
Cellular handling of receptor molecules is an important mechanism for
the regulation of appropriately-sensitive hormone-stimulated
signalling. 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 independent of its occupation with ligand. These
were also useful in western blotting and immunoprecipitation of this
receptor. Unstimulated CCK receptors remained on the surface of both
recombinant receptor-bearing 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
which were occupied with ligand. Unoccupied receptors remained on the
surface, uninfluenced by the stimulated signalling pathways.
Consistent with this, CCK receptor phosphorylation induced by 12-O
-tetradecanoyl-phorbol-13-acetate treatment did not stimulate receptor
internalization. After internalization, substantial receptor
recycling to the plasma membrane was observed. 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 signalling
event.
Received 16 April 1996; accepted in final form 13 September 1996.
APS Manuscript Number G139-6.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 7 October 1996