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.

Atrial natriuretic peptide (ANP), 1 a member of the natriuretic peptide family that includes brain natriuretic peptide and C-type natriuretic peptide, plays a fundamental role in the regulation of blood pressure, plasma volume, and renal function (1, 2). Two distinct classes of ANP receptors, named clearance and guanylyl cyclase (GC) receptors, have been biochemically and functionally well characterized (1, 3). Clearance receptors of ANP, the most abundant class of the natriuretic peptide receptors, have a single transmembrane domain, a short cytoplasmic tail of 37 amino acids, and an extracellular binding domain that has a significant homology to the extracellular domain of GC receptors (1, 4 -6). An extensive series of physiological, pharmacological, cellular, and genetic studies have shown that clearance receptors are importantly involved in the systemic and local clearance of ANP (7-11). This clearance function is accomplished by an efficient mechanism of receptor-mediated endocytosis. Endocytosed ANP is delivered to lysosomes, where it is hydrolyzed to its constituent amino acids, and the internalized receptors are recycled to the cell membrane (10, 12). The efficiency of this receptor-mediated endocytic mechanism is enhanced by a relatively low rate of dissociation of ANP from cell-surface receptors (12).Guanylyl cyclase subtype A (GCA) receptors mediate all of the known cardiovascular and renal effects of ANP (2, 13). GCA receptors have a single transmembrane domain, an extracellular ligand-binding domain, and a cytoplasmic domain constituted by a catalytic GC sequence and a tyrosine kinase-like (TK) sequence interposed between the transmembrane and the catalytic domains (14). Between the TK and GC sequences there is an amphipathic ␣-helical region that is involved in higher order oligomerization of GCA receptors (15,16). Under basal conditions, the TK domain has an inhibitory effect on GC activity. It has been postulated that upon ANP (or brain natriuretic peptide) binding to the extracellular domain, ATP binds to the TK domain and allosterically activates the catalytic GC domain (14,17).Previous studies in our laboratory have demonstrated that the native GCA in cultured glomerular mesangial and renomedullary interstitial cells is a constitutive membrane resident protein that does not undergo endocytosis and does not mediate lysosomal hydrolysis of ligand (18). Moreover, the dissociation of ANP from native GCA is very slow at subphysiological temperatures and increases exponentially at near physiological temperatures (18). We postulated that the rapid dissociation of ANP from surface GCA

ince the discovery of atrial natriuretic factor (ANF or ANP) in 1981 by De Bold et al. (l), S more than 6500 articles on the subject have appeared in the literature. For an extensive review on ANP and its receptors, the reader is directed to Reference 2. In this short review, we will consider in a noninclusive manner the dynamics and functional properties of natriuretic peptide receptors. ANP is a 28-amino acid polypeptide hormone secreted mainly by the heart atria in response to atrial stretch that results from an increase in central venous pressure, or systemic and pulmonary hypertension. ANP is a member of a family of natriuretic peptides, which include B-type or brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin. The physiological role of BNP, CNP, and urodilatin remains essentially unknown.The main actions of ANP in the mammalian organism are geared toward the regulation of volumepressure homeostasis (2,3). In the kidney, ANP exerts hemodynamic and tubular actions that lead to diuresis and natriuresis. In the cardiovascular system, ANP acts as a strong antagonist of vasoconstriction and also decreases plasma volume, a combination of events that reduces blood pressure. The decrease in plasma volume is due to a shift of fluid from the intravascular to the interstitial compartment brought about by an increase in hydraulic permeability of systemic capillaries. ANP is also a powerful functional antagonist of the renin-angiotensin-aldosterone system through inhibiting renin secretion by the kidney, aldosterone synthesis by the adrenal, and by antagonizing all known peripheral actions of angiotensin 11. The inhibition of the activity and effects of renin-angiotensinaldosterone contributes substantially to the cardiovas-' To whom requests for reprints should be addressed