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 i ntracellular trafficking of organelles that can be interrupted by competiti ve 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 re cycling of this receptor are well-established (J Cell Biol 128: 1029-1042, 1995), any influence of distinct receptor domains is not understood. In thi s work, constructs representing each of the intracellular domains of the CC K 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, interna lization, and desensitization. Three independent cell lines that coexpresse d the 25-residue second intracellular loop domain exhibited deficient resen sitization. In morphological assessment of receptor trafficking, this const ruct was also shown to interfere with receptor recycling to the plasma memb rane. As a control, recycling of an unrelated G protein-coupled receptor wa s demonstrated to occur normally in this cell line. These observations sugg est that rather than representing passive cargo within an endosome, a recep tor can influence its own trafficking within the cell.