Molecular determinants of the clearance function of type C receptors of natriuretic peptides

Receptor-mediated endocytosis is the cellular mechanism by which type C receptors of natriuretic peptides exert their clearance function. In the present work, performed in recombinant Chinese hamster ovary cells stably transfected with wild type or mutated human kidney C receptors we determined net endocytic rates (ER) of C receptor-ligand complexes, lysosomal hydrolysis of ligand (I-125-labeled native atrial natriuretic factor, ANF(1-28)), and receptor recycling, Equilibrium ligand binding, immunocytochemistry, and immunoprecipitation were performed to characterize the transfected receptors, The net ER of recombinant wild type C receptors was similar to 6% of occupied receptors internalized per min, and C receptor-mediated lysosomal hydrolysis of ligand amounted to similar to 250% of specifically bound I-125-ANF(1-28)/h, with efficient recycling of internalized C receptors to the cell surface. Hypertonic sucrose reduced net ER and lysosomal hydrolysis of I-125-ANF(1-28) more than 10-fold, indicating that endocytosis occurred via clathrin-coated pits. Total deletion of the cytoplasmic domain also reduced net ER and lysosomal hydrolysis of I-125-ANF(1-28) by almost 10-fold whereas deletion of the terminal 28 amino acids of the cytoplasmic tail led to a 4-fold reduction in these parameters, Replacement of cytoplasmic domain Tyr(508) by Ala, or Tyr(508) and Phe(538) by Ala, reduced net endocytosis and lysosomal hydrolysis of I-125-ANF(1-28) by 40-50%. Replacement of extracellular domain Cys(473) by Ala impeded the constitutive formation of homodimers and reduced by similar to 50% the net ER and lysosomal hydrolysis of I-125-ANF(1-28). These results demonstrate that the cytoplasmic domain of C receptors, Tyr(508) within this domain, and constitutive receptor dimerization are the major molecular determinants of the clearance function of C receptors.