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 (125I-labeled native atrial natriuretic factor, ANF1-28), 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 approximately 6% of occupied receptors internalized per min, and C receptor-mediated lysosomal hydrolysis of ligand amounted to approximately 250% of specifically bound 125I-ANF1-28/h, with efficient recycling of internalized C receptors to the cell surface. Hypertonic sucrose reduced net ER and lysosomal hydrolysis of 125I-ANF1-28 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 125I-ANF1-28 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 Tyr508 by Ala, or Tyr508 and Phe538 by Ala, reduced net endocytosis and lysosomal hydrolysis of 125I-ANF1-28 by 40-50%. Replacement of extracellular domain Cys473 by Ala impeded the constitutive formation of homodimers and reduced by approximately 50% the net ER and lysosomal hydrolysis of 125I-ANF1-28. These results demonstrate that the cytoplasmic domain of C receptors, Tyr508 within this domain, and constitutive receptor dimerization are the major molecular determinants of the clearance function of C receptors.
Cohen
D
D
Department of Physiology, Cornell University Medical College, New York, New York 10021, USA.
Koh
G Y
GY
Nikonova
L N
LN
Porter
J G
JG
Maack
T
T
D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D006162:Guanylate Cyclase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D017461:Receptors, Atrial Natriuretic Factor / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D009320:Atrial Natriuretic Factor; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C073616:atrial natriuretic factor receptor C