Abstract 282: Aromatic Residue-based Tetrapeptide Fqqi Motif Mediates Internalization and Trafficking of Guanylyl-cyclase/natriuretic Peptide Receptor-a in Mouse Mesangial Cells

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Abstract

Atrial natriuretic peptide (ANP) is a cardiac hormone that modulates blood pressure and blood volume by activation of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) and subsequent generation of intracellular second messenger cGMP. The aim of the present study was to examine the role of small sequence motifs in the internalization and trafficking pathway of NPRA by immunofluorescence confocal microscopy. A chimeric protein of enhanced green fluorescence protein (eGFP) and NPRA (eGFP-NPRA) was used to study the dynamics of NPRA trafficking in the intact mouse mesangial cells (MMCs). We have identified a FQQI (Phe790, Gln791, Gln792 and Ile793) motif in the carboxyl-terminal domain of NPRA sequence and mutated with alanine residues (FQQI/AQQA) using QuickChange II site-directed mutagenesis kit. Cells were transiently transfected with mutated and wild-type constructs and cultured at 37 0C in an atmosphere of 5% CO2 and 95% O2. In the ANP-treated cells, colocalization of wild-type eGFP-NPRA with pan-Cadherin, a plasma membrane marker, indicated that internalization of receptor accounted for 59.2%±3.4, 70.0%±4.3, 71.8%±4.3, and 77.4%±3.2 at 5, 10, 15, and 30 min, respectively of total fluorescence intensity compared with controls. Colocalization of eGFP-NPRA with early endosome antigen-1 (EEA-1) marker was maximum at 5 min (64.5%±2.3), and gradually decreased at 30 min (24.4%±3.8). However, the mutation of FQQI/AQQA decreased

the receptor internalization by 45% (P< 0.001) and the receptor colocalization in to endosome by 58% (P<0.0001), respectively, compared with wild type receptor. After ANP treatment, immunofluorescence analysis showed a marked increase in the accumulation of intracellular cGMP (29-fold) concurrent with receptor internalization compared with controls. The results indicate that ligand-receptor complexes continue to traffic and produce cGMP even after receptor sequestration into endosomes. Our, data demonstrate that FQQI motif plays essential role in the internalization of NPRA, which regulates hypertension and cardiovascular homeostasis.

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