Impaired resensitization and recycling of the cholecystokinin receptor by co-expression of its second intracellular loop.

Intermolecular interaction represents an important theme in regulation of intracellular trafficking of organelles that can be interrupted by competitive overexpression of a relevant molecular domain. We attempted to identify the functional importance of intracellular domains of the cholecystokinin (CCK) receptor by their over-expression in receptor-bearing Chinese hamster ovary (CHO-CCKR) cell lines. Although clathrin-dependent endocytosis and recycling of this receptor are well-established (J Cell Biol 128:1029-1042, 1995), any influence of distinct receptor domains is not understood. In this work, constructs representing each of the intracellular domains of the CCK receptor were coexpressed with wild-type receptor, and stable clonal cell lines were selected. Each was characterized for ligand binding and agonist-stimulated biological activity (inositol 1,4,5-trisphosphate generation), desensitization, resensitization, receptor internalization, and recycling. Each cell line expressed normal CCK radioligand binding, signaling, internalization, and desensitization. Three independent cell lines that coexpressed the 25-residue second intracellular loop domain exhibited deficient resensitization. In morphological assessment of receptor trafficking, this construct was also shown to interfere with receptor recycling to the plasma membrane. As a control, recycling of an unrelated G protein-coupled receptor was demonstrated to occur normally in this cell line. These observations suggest that rather than representing passive cargo within an endosome, a receptor can influence its own trafficking within the cell.