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Rapid Internalization and Trafficking of GC‐A/NPRA via Endo‐lysosomal Compartments with Concurrent Generation of cGMP in Mouse Mesangial Cells: Role of FQQI Motif
Author(s) -
Mani Indra,
Garg Renu,
Tripathi Satyabha,
Pandey Kailash N
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.967.19
Subject(s) - internalization , endosome , microbiology and biotechnology , endocytic cycle , clathrin , signal transducing adaptor protein , biology , green fluorescent protein , receptor , colocalization , immunoprecipitation , immunofluorescence , chemistry , endocytosis , signal transduction , intracellular , cell culture , biochemistry , antibody , genetics , gene , immunology
The objective of this study was to delineate the critical role of endocytic signal in intracellular sorting and signaling of receptor into these compartments. We have identified a FQQI (Phe 790 , Gln 791 , Gln 792 and Ile 793 ) motif in the cytoplasmic tail of NPRA sequence and mutated with alanine residues (FQQI/AAAA) using QuickChange II site‐directed mutagenesis kit in enhanced green fluorescence protein (eGFP)‐tagged NPRA (eGFP‐NPRA) cDNA sequence. Mouse mesangial cells (MMCs) were transiently transfected with both mutant and wild‐type constructs and cultured at 37 0 C in an atmosphere of 5% CO 2 and 95% O 2 . We utilized immunofluorescence staining and co‐immunoprecipitation (co‐IP) of plasma membrane, adaptor protein‐1, endosomal, lysosomal, and Rab 11 markers to follow subcellular trafficking and signaling by confocal immunofluorescence microscopy and immunoblotting. The treatment of cells with ANP at different times accelerated the internalization of receptor from cell surface to cell interior. The internalization studies revealed the immunofluorescence colocalization of mutated motif (FQQI/AAAA) receptor with pan‐Cadherin, a plasma membrane marker, and showed a decreased internalization of eGFP‐NPRA by 49%±3.4 ( P< 0.001) compared with the wild‐type receptor. Interestingly, we show that μ 1 B subunit of adaptor protein‐1 (AP‐1, μ 1 B), binds directly to a phenylalanine based FQQI motif in the cytoplasmic tail of receptor. However, subcellular trafficking studies indicated that colocalization of mutated receptor with μ 1 B of AP‐1, early endosome antigen‐1 (EEA‐1), lysosome‐associated membrane protein‐1 (LAMP‐1), and Rab 11 marker, was decreased by 50%±3.2 ( P< 0.001), into early endosome by 57%±4.7 ( P< 0.001), lysosome by 32%±1.4 ( P< 0.01), and recycling endosome by 40%±1.8 ( P< 0.01) respectively, compared with wild‐type receptor. The receptor containing mutated motif (FQQI/AAAA) also produced a significantly decreased level 64%±3.6 ( P< 0.001) intracellular cGMP during subcellular trafficking compared to WT receptor. The co‐immunoprecipitation assay confirmed a decreased level of immunofluorescence colocalization of mutant receptor with subcellular compartments during endocytic processes. The results suggest that Phe 790 and Ile 793 constitute important element in FQQI motif, which are essential for the internalization and trafficking of NPRA. The findings may provide the basis for new molecular association of single nucleotide polymorphisms in Npr1 gene with susceptibility to diseases and /or resistance to drugs in translational studies. Support or Funding Information This work was supported by NIH grants R01HL057531 and R01HL062147