Binding of 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 endocytic signal in intracellular sorting of the receptor, we have identified a FQQI (Phe⁷⁹⁰, Gln⁷⁹¹, Gln⁷⁹², and Ile⁷⁹³) 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 eGFP-NPRA cDNA sequence, markedly attenuated the internalization of mutant receptors by almost 49% compared with the WT receptor. Interestingly, we show that μ1B subunit of adaptor protein-1 (AP-1, μ1B), binds directly to a phenylalanine based FQQI motif in the cytoplasmic tail of receptor. However, subcellular trafficking indicated that immunofluorescence colocalization of mutated receptor with early endosome antigen-1 (EEA-1), lysosome-associated membrane protein-1 (LAMP-1), and Rab 11 marker, was decreased by 57% into early endosome, 48% in lysosome, and 42% in recycling endosome, respectively, compared with WT receptor in MMCs. The receptor containing mutated motif (FQQI/AAAA) also produced a significantly decreased level of intracellular cGMP during subcellular trafficking than WT receptor. The co-immunoprecipitation assay confirmed a decreased level of colocalization of mutant receptor with subcellular compartments during endocytic processes. The results suggest that FQQI motif is essential for the internalization and subcellular trafficking of NPRA during the hormone signaling process in intact MMCs.