Differential internalization and processing of atrial-natriuretic-factor B and C receptor in PC12 cells

Abstract

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.

Neuronal Ca2+ sensor protein VILIP-1 affects cGMP signalling of guanylyl cyclase B by regulating clathrin-dependent receptor recycling in hippocampal neurons

“…These results fit to the kinetics of the VILIP-1 effect on GC activity (this study) and the observed receptor half-life during desensitization of the related receptor GC-A (Pandey et al, 2002). Moreover, a very similar effect of monesin on the recycling of GC-A, where monensin blocked the reappearance of internalized receptors on the cell surface, was described in PC12 cells (Rathinavelu and Isom, 1991).…”

“…It has been previously shown that a desensitization-dependent internalization of GC-A occurs (Rathinavelu and Isom, 1991) and that the reduction of surface expression of GC-A reaches a maximum level approximately 15-20 minutes after receptor stimulation (Pandey et al, 2002). By contrast, at this time point the receptor response is not desensitized in VILIP-1-transfected cells, but a rather strong increase in cGMP accumulation can be noticed compared with non-transfected cells (Fig.…”

Characterization and partial purification of the human receptor for the heat‐stable enterotoxin

“…However, in the ligand-binding domain, very little similarity is observed, and this is reflected in the lack of interaction of ligands amongst various members of the guanylyl cyclase receptor family. The pH-dependent binding that we report in this study for the ST receptor is in direct contrast to the atrial natriuretic factor receptor where low pH actually induces rapid dissociation of the ligand [38]. When one considers the marginally acidic microenvironment of the small intestine, it is apparent that ST has exploited this in its interactions with the receptor.…”

Characterization of natriuretic peptide receptors in cultured cells.

“…510 " 13 Two distinct types of receptors have been identified. One type of receptor is a particulate guanylate cyclase 14 and is designated as the biologically active receptor.…”