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.
Pandey
Kailash N
KN
Department of Physiology, Tulane University Health Sciences Center and School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112, USA. kpandey@tulane.edu
Nguyen
Huong T
HT
Garg
Renu
R
Khurana
Madan L
ML
Fink
Jude
J
D020816:Amino Acid Motifs; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D021381:Protein Transport; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry*
D014443:Tyrosine; D009320:Atrial Natriuretic Factor; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A