DIFFERENTIAL INTERNALIZATION AND PROCESSING OF ATRIAL-NATRIURETIC-FACTOR B-RECEPTORS AND C-RECEPTORS 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 I-125-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 I-125-ANF(99-126) was reduced by 24 %, indicating B receptors mediate 76 % of ligand internalization. Incubation of cells with 10-mu-M-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 I-125-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.