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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.

A hybrid gene was constructed containing a fusion between the DNA sequences encoding the secretory precursor of the yeast mating pheromone alpha-factor and a synthetic sequence encoding a biologically active 24-amino acid carboxyl-terminal portion of the human atrial natriuretic peptide (hANP) precursor. Transformation of Saccharomyces cerevisiae with the hybrid gene resulted in the yeast cells secreting biologically active hANP into the extracellular medium. The secreted hANP was purified and found to be accurately processed at the junction in the chimeric alpha-factor/hANP protein, producing the desired mature hANP amino terminus. The secreted product was also folded correctly with respect to the single disulfide bond. However, the carboxyl terminus of the secreted hANP material was heterogeneous such that the major form lacked the last two amino acids of the peptide while the minor form was the full length material. The observed processing at the carboxyl terminus of the secreted hANP may reflect a normal processing event involved in alpha-factor peptide maturation.

Adsorptive and/or receptor-mediated endocytosis of proteins is a universal cell property, which is highly expressed in epithelial cells. Some absorbed proteins are transported intact across cells and in this manner subserve specialized functions such as the transference of immunity from mother to child. Mainly, however, absorbed proteins are transported to lysosomes, where they undergo complete hydrolysis to amino acids. This process is essential for the homeostasis of circulating proteins. This brief review considers the intracellular pathways taken by endocytosed proteins and the quantitative aspects and determinants of protein uptake and catabolism by epithelial cells. The topics to be briefly discussed are initial internalization sites, transport organelles (endosomes), and lysosomal and nonlysosomal pathways of transport; intracellular sorting of internalized proteins, membranes, and receptors; kinetics and selectivity of renal cell uptake of low-molecular-weight proteins and proteohormones; receptor-mediated endocytosis of larger proteins (e.g., glycoproteins) by hepatocytes; and lysosomal catabolism of absorbed proteins and its dependence on protein load and endosomal-lysosomal pH and function. The perspectives of the field and some of the outstanding unsolved problems are briefly discussed.

The mechanism of tubular uptake of labeled human growth hormone ([125I]hGH), a low molecular weight protein (approximate 21,5000 daltons), was studied in isolated perfused rat kidneys. Fractional reabsorption (FR) of [125I]hGH was decreased from 94 to 77% over a period of 80 min as perfusate oncotic pressure was lowered by reducing the albumin concentration from 7.5 to 2.5 g/100ml, whereas greater reductions in fractional sodium (delta 35%) and fluid reabsorption (delta 42%) occurred, indicating that tubular [125I]hGH uptake is likely a specific process not directly dependent upon net fluid and sodium reabsorption. Absolute absorption rates of [125I]hGH filtered loads were inhibited by cytochalasin B, a microfilament disrupter when kidneys were perfused with either albumin concentration. Cytochalasin B inhibited [125I]hGH absorption in both a dose-and time-related manner. The low dose of cytochalasin B (2.5 micrograms/ml) decreased [125I]hGH absorption without significantly altering sodium, fluid or glucose reabsorption. With high doses (5 and 10 micrograms/ml), cytochalasin B affected tubular absorption of [125I]hGH to an extent much greater than sodium, fluid and glucose reabsorption. Inhibition of cytochalasin B on FR[125I]hGH was poorly correlated with the concurrent inhibition of FRNa and FRH2O. Accordingly, tubular reabsorption of [125I]hGH is not directly linked to that of sodium, fluid and glucose. The present studies are consistent with the hypothesis that renal absorption of low molecular-weight proteins is via an endocytotic process involving microfilaments.