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 (Δ1045-Npra) affected neither ligand-binding efficiency nor cGMP production. However, deletion of 120 to 170 aa residues (Δ937-Npra, Δ916-Npra, Δ902-Npra, and Δ887-Npra) decreased ligand binding by 16 to 20% and cGMP production by 50 to 90%. Further deletion of 422 aa and 569 aa (Δ635-Npra and Δ488-Npra) reduced ligand binding efficiency by 40% and 90%, respectively. The deletion of 12 aa (Δ1045-Npra) did not affect the internalization of Npra; however, deletions up to 170 aa (Δ937-Npra, Δ916-Npra, Δ887-Npra) reduced the internalization of ligand-receptor complexes by 60%. Cells expressing either full-length (wild-type) Npra or 120 aa deleted receptor (Δ937-Npra) released 40 to 45% ¹²⁵I-ANP radioactivity into culture medium, but only 10 to 15% radioactivity was released from the cells that expressed Δ635-Npra. Furthermore, 35 to 40% ¹²⁵I-ANP radioactivity was detected into the intracellular compartments of cells that expressed the wild-type Npra, and only 5 to 10% ¹²⁵I-ANP radioactivity was observed in cells expressing the Δ635-Npra (−422 aa) or Δ488-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.