title: Antibody tracking demonstrates cell type-specific and ligand-independent internalization of guanylyl cyclase a and natriuretic peptide receptor C.
abstract: Atrial natriuretic peptide (ANP) binds guanylyl cyclase-A (GC-A) and natriuretic peptide receptor-C (NPR-C). Internalization of GC-A and NPR-C is poorly understood, in part, because previous studies used (125)I-ANP binding to track these receptors, which are expressed in the same cell. Here, we evaluated GC-A and NPR-C internalization using traditional and novel approaches. Although HeLa cells endogenously express GC-A, (125)I-ANP binding and cross-linking studies only detected NPR-C, raising the possibility that past studies ascribed NPR-C-mediated processes to GC-A. To specifically measure internalization of a single receptor, we developed an (125)I-IgG-binding assay that tracks extracellular FLAG-tagged versions of GC-A and NPR-C independently of each other and ligand for the first time. FLAG-GC-A bound ANP identically with wild-type GC-A and was internalized slowly (0.5%/min), whereas FLAG-NPR-C was internalized rapidly (2.5%/min) in HeLa cells. In 293 cells, (125)I-ANP and (125)I-IgG uptake curves were superimposable because these cells only express a single ANP receptor. Basal internalization of both receptors was 8-fold higher in 293 compared with HeLa cells and ANP did not increase internalization of FLAG-GC-A. For FLAG-NPR-C, neither ANP, BNP, nor CNP increased its internalization in either cell line. Prolonged ANP exposure concomitantly reduced surface and total GC-A levels, consistent with rapid exchange of extracellular and intracellular receptor pools. We conclude that ligand binding does not stimulate natriuretic peptide receptor internalization and that cellular environment determines the rate of this process. We further deduce that NPR-C is internalized faster than GC-A and that increased internalization is not required for GC-A down-regulation.
authors.affiliation: Department of Biochemistry, University of Minnesota—Twin Cities, Minneapolis, Minnesota 55455, USA.
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Dickey
authors.forename: Darcy R
authors.initials: DR
authors.lastname: Flora
authors.forename: Lincoln R
authors.initials: LR
authors.lastname: Potter
mesh_terms: D000906:Antibodies / Q000276:immunology*; D006162:Guanylate Cyclase / Q000276:immunology*; D006367:HeLa Cells; D006801:Humans; D007457:Iodine Radioisotopes; D008024:Ligands; D010957:Plasmids; D017461:Receptors, Atrial Natriuretic Factor / Q000276:immunology*
chemical_list: D000906:Antibodies; D007457:Iodine Radioisotopes; D008024:Ligands; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor