TY - JOUR
T1 - Impaired resensitization and recycling of the cholecystokinin receptor by co-expression of its second intracellular loop
AU - Ding, Xi Qin
AU - Rao, Rammohan V.
AU - Kuntz, Susan M.
AU - Holicky, Eileen L.
AU - Miller, Laurence J.
PY - 2000
Y1 - 2000
N2 - 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.
AB - 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.
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U2 - 10.1124/mol.58.6.1424
DO - 10.1124/mol.58.6.1424
M3 - Short survey
C2 - 11093782
AN - SCOPUS:0033669729
VL - 58
SP - 1424
EP - 1433
JO - Molecular Pharmacology
JF - Molecular Pharmacology
SN - 0026-895X
IS - 6
ER -