Fig. 1.

Schematic representation of the intracellular trafficking motif, expression, and amino acid residue requirements (AAAA, AQQA, AQQI, and FQQA) for internalization of wild-type (WT) and mutant (natriuretic peptide receptor-A; NPRA) in intact mouse mesangial cells (MMCs). A: topology indicating the extracellular, transmembrane (TM), and cytoplasmic regions of NPRA. The internalization motif analyzed corresponds to amino acids 790–793. eGFP, enhanced green fluorescent protein. B: amino acid sequence of the WT internalization motif in the carboxyl-terminal domain of NPRA as indicated by the single letter code. Δ790–793 indicates the internal substitution of residues Phe⁷⁹⁰, Gln⁷⁹¹, Gln⁷⁹², and Ile⁷⁹³ with alanine. C: lanes a, b, c, d, and e represents the expressed WT and mutant receptor (AAAA, AQQA, AQQI, and FQQA) bands in MMCs, respectively. The arrow indicates the position of 162-kDa eGFP-NPRA fusion protein band. D: alanine substitutions at amino acid positions 788–795 and in different combinations in the FQQI motif. Confluent MMCs in 6-cm² dishes transiently transfected with WT and mutant receptors were pretreated with 100 nM C-atrial natriuretic factor receptor ligand (C-ANF) to block the NPRC, and then treated with ¹²⁵I-labeled atrial natriuretic peptide (ANP) at 4°C for 1 h in the absence or presence of unlabeled ANP. After 15 min of incubation, the internalization of ligand-receptor complexes was quantified as described in materials and methods. Values are means ± SE of 6 separate experiments in triplicate. **P < 0.01, ***P < 0.001 relative to WT receptor.