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.
Ding
X Q
XQ
Center for Basic Research in Digestive Diseases, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Rao
R V
RV
Kuntz
S M
SM
Holicky
E L
EL
Miller
L J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005455:Fluorescent Antibody Technique; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000502:physiology; D011487:Protein Conformation; D011949:Receptors, Cholecystokinin / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
D008565:Membrane Proteins; D010446:Peptide Fragments; D011949:Receptors, Cholecystokinin