A novel cytoplasmic tail fqqi motif mediates internalization and intracellular trafficking of guanylyl‐cyclase/natriuretic peptide receptor‐A (539.4)

Abstract

Cardiac hormone, atrial natriuretic peptide (ANP) modulates blood pressure and blood volume by activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which produces the second messenger cGMP. The aim of the present study was to examine the role of small sequence motifs in the internalization and intracellular 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 NPRA trafficking in the intact mouse mesangial cells (MMCs). We have identified a FQQI (Phe790, Gln791, Gln792 and Ile793) motif in the cytoplasmic tail of NPRA sequence and mutated with alanine residues (FQQI/AQQA) and cells were transiently transfected with both constructs. 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%, 70.0%, 71.8%, and 77.4% at 5, 10, 15, and 30 min, respectively of total fluorescence intensity compared with untreated cells. Colocalization of eGFP‐NPRA with early endosome antigen‐1 (EEA‐1) marker was maximum at 5 min (64.5%), and gradually decreased at 30 min (24.4%). However, the mutation of FQQI/AQQA decreased the receptor internalization by 45% (P< 0.001) and the receptor colocalization into endosome by 58% (P<0.001), respectively, compared with wild type receptor. Our, study suggest that a novel FQQI motif in the cytoplasmic tail mediates the internalization and intracellular trafficking of NPRA, which plays critical roles in hypertension and cardiovascular homeostasis.Grant Funding Source: Supported by the NIH grant (HL57531)