title: Functional domains and expression of truncated atrial natriuretic peptide receptor-A: the carboxyl-terminal regions direct the receptor internalization and sequestration in COS-7 cells.
abstract: The objective of this study was to determine the role of cytoplasmic (protein kinase-like homology and guanylyl cyclase catalytic) domains of atrial natriuretic peptide (ANP) receptor-A (Npra) in postbinding events and metabolic turnover of ligand-receptor complexes. Using deletion mutagenesis, the specific regions in the intracellular domains of Npra relevant to the receptor function, namely ligand-binding, cGMP production, and internalization and sequestration of ligand-receptor complexes, have been determined in transiently expressing COS-7 cells. Deletion of 12 aa (aa) at the carboxyl-terminal end of receptor (Delta1045-Npra) affected neither ligand-binding efficiency nor cGMP production. However, deletion of 120 to 170 aa residues (Delta937-Npra, Delta916-Npra, Delta902-Npra, and Delta887-Npra) decreased ligand binding by 16 to 20% and cGMP production by 50 to 90%. Further deletion of 422 aa and 569 aa (Delta635-Npra and Delta488-Npra) reduced ligand binding efficiency by 40% and 90%, respectively. The deletion of 12 aa (Delta1045-Npra) did not affect the internalization of Npra; however, deletions up to 170 aa (Delta937-Npra, Delta916-Npra, Delta887-Npra) reduced the internalization of ligand-receptor complexes by 60%. Cells expressing either full-length (wild-type) Npra or 120 aa deleted receptor (Delta937-Npra) released 40 to 45% (125)I-ANP radioactivity into culture medium, but only 10 to 15% radioactivity was released from the cells that expressed Delta635-Npra. Furthermore, 35 to 40% (125)I-ANP radioactivity was detected into the intracellular compartments of cells that expressed the wild-type Npra, and only 5 to 10% (125)I-ANP radioactivity was observed in cells expressing the Delta635-Npra (-422 aa) or Delta488-Npra (-569 aa) mutant receptors. These results show that specific regions within the intracellular domains of Npra determine the extent of ligand-binding efficiency, cGMP production, endocytosis, and intracellular sequestration of ligand-receptor complexes in cDNA expressing COS-7 cells.
authors.affiliation: Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana, USA. kpandey@mailhost.tcs.tulane.edu
authors.forename: K N
authors.initials: KN
authors.lastname: Pandey
authors.forename: R
authors.initials: R
authors.lastname: Kumar
authors.forename: M
authors.initials: M
authors.lastname: Li
authors.forename: H
authors.initials: H
authors.lastname: Nguyen
mesh_terms: D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D020134:Catalytic Domain; D006152:Cyclic GMP / Q000378:metabolism; D017353:Gene Deletion; D006162:Guanylate Cyclase / Q000378:metabolism; D007457:Iodine Radioisotopes; D051379:Mice; D011487:Protein Conformation; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015290:Second Messenger Systems
chemical_list: D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; D006152:Cyclic GMP