Par-4 inhibits choline uptake by interacting with CHT1 and reducing its incorporation on the plasma membrane.
CHT1 is a Na(+)- and Cl(-)-dependent, hemicholinium-3 (HC-3)-sensitive, high affinity choline transporter. Par-4 (prostate apoptosis response-4) is a leucine zipper protein involved in neuronal degeneration and cholinergic signaling in Alzheimer's disease. We now report that Par-4 is a negative regulator of CHT1 choline uptake activity. Transfection of neural IMR-32 cells with human CHT1 conferred Na(+)-dependent, HC-3-sensitive choline uptake that was effectively inhibited by cotransfection of Par-4. Mapping studies indicated that the C-terminal half of Par-4 was physically involved in interacting with CHT1, and the absence of Par-4.CHT1 complex formation precluded the loss of CHT1-mediated choline uptake induced by Par-4, indicating that Par-4.CHT1 complex formation is essential. Kinetic and cell-surface biotinylation assays showed that Par-4 inhibited CHT1-mediated choline uptake by reducing CHT1 expression in the plasma membrane without significantly altering the affinity of CHT1 for choline or HC-3. These results suggest that Par-4 is directly involved in regulating choline uptake by interacting with CHT1 and by reducing its incorporation on the cell surface.
Xie
Jun
J
Department of Physiology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Boulevard, Oklahoma City, OK 73104, USA.
Guo
Qing
Q
D000818:Animals; D051017:Apoptosis Regulatory Proteins; D019921:Biotinylation; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D016923:Cell Death; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002794:Choline / Q000378:metabolism* / Q000494:pharmacology; D018678:Cholinergic Agents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006426:Hemicholinium 3 / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D018613:Microscopy, Confocal; D009447:Neuroblastoma / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D010641:Phenotype; D011233:Precipitin Tests; D016548:Prosencephalon / Q000166:cytology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014162:Transfection
D051017:Apoptosis Regulatory Proteins; C488255:CHT1 protein, mouse; D002352:Carrier Proteins; D018678:Cholinergic Agents; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D026901:Membrane Transport Proteins; C065637:choline transporter; C101296:prostate apoptosis response-4 protein; D006426:Hemicholinium 3; D002794:Choline