Differential internalization and processing of atrial-natriuretic-factor B and C receptor in PC12 cells.
PC12 cells express two atrial-natriuretic-factor-(ANF)-receptor subtypes with molecular masses of 130,000 (B receptor) and 70,000 (C receptor). The B-receptor subtype constitutes 65% of the cell-surface receptor population, and the remaining 35% are C receptors as determined by saturation binding studies in the presence of C-ANF, a C-receptor-selective analogue. ANF-(99-126)-peptide [ANF(99-126)], which can bind to both B- and C-receptor subtypes, was rapidly internalized into the cells after incubation at 37 degrees C. Internalization of 125I-ANF(99-126) was used as an index of the receptor-mediated endocytosis and to quantify receptor internalization. In the presence of a saturating concentration of C-ANF, receptor-mediated internalization of 125I-ANF(99-126) was reduced by 24%, indicating B receptor mediate 76% of ligand internalization. Incubation of cells with 10 microM-ANF at 37 degrees C down-regulated both receptor subtypes as reflected by decreased surface binding. Time-dependent studies suggest that B- and C-receptor subtypes undergo differential down-regulation. Incubation of down-regulated cells for 120 min in ANF-free medium produced a recovery of 35% of the original cell-surface binding. Affinity cross-linking of 125I-ANF to the receptors on the plasma membrane in re-incubated (up-regulated) cells demonstrated expression of predominantly the B-receptor subtype. Monensin blocked 72% of receptor up-regulation, whereas cycloheximide inhibited 43%, suggesting an active recycling mechanism involved in mediating up-regulation of the B receptors. The present study demonstrates a rapid internalization and intracellular recycling mechanism for B receptors in PC12 cells. C receptors also undergo internalization and down-regulation, but recycling of this receptor subtype into the plasma membrane occurs at a lower rate and to a lesser extent than is the case for the B receptor.
Rathinavelu
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Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907.
Isom
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D000310:Adrenal Gland Neoplasms; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D015536:Down-Regulation; D007700:Kinetics; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010673:Pheochromocytoma; D051381:Rats; D017461:Receptors, Atrial Natriuretic Factor; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D003571:Cytochalasin B; D009320:Atrial Natriuretic Factor; D008985:Monensin; D003513:Cycloheximide; D017461:Receptors, Atrial Natriuretic Factor