Internalization and trafficking of guanylyl (guanylate) cyclase/natriuretic peptide receptor A is regulated by an acidic tyrosine-based cytoplasmic motif GDAY

We have identified a GDAY motif in the C-terminal domain of guanylyl cyclase (guanylate cyclase)/NPRA (natriuretic peptide receptor A) sequence, which serves a dual role as an internalization signal and a recycling signal. To delineate the role of the GDAY motif in receptor internalization and sequestration, we mutated Gly⁹²⁰, Asp⁹²¹ and Tyr⁹²³ to alanine residues (GDAY/AAAA) in the NPRA cDNA sequence. The cDNAs encoding wild-type and mutant receptors were transfected in HEK-293 cells (human embryonic kidney 293 cells). The internalization studies of ligand–receptor complexes revealed that endocytosis of ¹²⁵I-ANP by HEK-293 cells expressing G920A, Y923A or GDAY/AAAA mutant receptor was decreased by almost 50% (P<0.001) when compared with cells expressing the wild-type receptor. However, the effect of D921A mutation on receptor internalization was minimal. Ligand-mediated down-regulation of G920A, Y923A and GDAY/AAAA mutant receptors was decreased by 35–40% when compared with wild-type NPRA. Subsequently, the recycling of internalized D921A and GDAY/AAAA mutant receptors from the intracellular pool was decreased by more than 40±4% when compared with wild-type NPRA. Recycling of G920A and Y923A mutant receptors was also decreased, but to a significantly lesser extent compared with the D921A or GDAY/AAAA mutant receptors. We conclude that the Gly⁹²⁰ and Tyr⁹²³ residues within the GDAY consensus motif are necessary for internalization, and that residue Asp⁹²¹ is important for recycling of NPRA. The current results provide new evidence for a dual role of the GDAY sequence motif in ligand-mediated internalization, recycling and down-regulation of a single-transmembrane receptor protein NPRA.