title: Role of FQQI motif in the internalization, trafficking, and signaling of guanylyl-cyclase/natriuretic peptide receptor-A in cultured murine mesangial cells.
abstract: Binding of the cardiac hormone atrial natriuretic peptide (ANP) to transmembrane guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), produces the intracellular second messenger cGMP in target cells. To delineate the critical role of an endocytic signal in intracellular sorting of the receptor, we have identified a FQQI (Phe(790), Gln(791), Gln(792), and Ile(793)) motif in the carboxyl-terminal region of NPRA. Mouse mesangial cells (MMCs) were transiently transfected with the enhanced green fluorescence protein (eGFP)-tagged wild-type (WT) and mutant constructs of eGFP-NPRA. The mutation FQQI/AAAA, in the eGFP-NPRA cDNA sequence, markedly attenuated the internalization of mutant receptors by almost 49% compared with the WT receptor. Interestingly, we show that the μ1B subunit of adaptor protein-1 binds directly to a phenylalanine-based FQQI motif in the cytoplasmic tail of the receptor. However, subcellular trafficking indicated that immunofluorescence colocalization of the mutated receptor with early endosome antigen-1 (EEA-1), lysosome-associated membrane protein-1 (LAMP-1), and Rab 11 marker was decreased by 57% in early endosomes, 48% in lysosomes, and 42% in recycling endosomes, respectively, compared with the WT receptor in MMCs. The receptor containing the mutated motif (FQQI/AAAA) also produced a significantly decreased level of intracellular cGMP during subcellular trafficking than the WT receptor. The coimmunoprecipitation assay confirmed a decreased level of colocalization of the mutant receptor with subcellular compartments during endocytic processes. The results suggest that the FQQI motif is essential for the internalization and subcellular trafficking of NPRA during the hormone signaling process in intact MMCs.
authors.affiliation: Department of Physiology, Tulane University Health Sciences Center and School of Medicine, New Orleans, Louisiana.
authors.forename: Indra
authors.initials: I
authors.lastname: Mani
authors.affiliation: Department of Physiology, Tulane University Health Sciences Center and School of Medicine, New Orleans, Louisiana.
authors.forename: Renu
authors.initials: R
authors.lastname: Garg
authors.affiliation: Department of Physiology, Tulane University Health Sciences Center and School of Medicine, New Orleans, Louisiana kpandey@tulane.edu.
authors.forename: Kailash N
authors.initials: KN
authors.lastname: Pandey
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D050527:Mesangial Cells / Q000201:enzymology*; D051379:Mice; D009154:Mutation; D021381:Protein Transport; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015290:Second Messenger Systems; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D006152:Cyclic GMP; D049452:Green Fluorescent Proteins; D051907:Lysosomal Membrane Proteins; D017461:Receptors, Atrial Natriuretic Factor; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C052255:Lamp1 protein, mouse; D052118:Lysosomal-Associated Membrane Protein 1; C093846:early endosome antigen 1; C485184:enhanced green fluorescent protein; C091715:atrial natriuretic factor receptor A
keywords: guanylyl cyclase/natriuretic peptide receptor-A; immunofluorescence; mouse mesangial cells; receptor internalization; trafficking