0031-399819513806-0835$03.0010 PEDIATRIC RESEARCH Vol. 38, No. 6, 1995 Copyright O 1995 International Pediatric Research Foundation, Inc. Printed in U.S.A. Receptor Cell Biology: Receptor-Mediated Endocytosis ALAN L. SCHWARTZ Departments of Pediatrics, Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 ACT Receptor-mediated endocytosis (RME) provides one major Abbreviations pathway for the trafficking of extracellular molecules into the RME, receptor-mediated endocytosis cell. This involves the binding of a ligand to a specific cell ASGP, asialoglycoprotein surface receptor, clustering of the ligand-receptor complexes in EGF, epidermal growth factor coated pits, invagination and pinching off of the coated pits to AP, adaptins form coated vesicles, and delivery of coated vesicles to discrete CURL, compartment of uncoupling receptor and ligand membrane-limited cytoplasmic sorting organelles, endosomes. Man-6-P, mannose 6-phosphate Within these endosomes, ligands and receptors are each targeted t-PA, tissue-type plasminogen activator to their appropriate cellular destination (e.g., lysosome, cyto- u-PA, urokinase plasminogen activator plasminogen activator plasm, opposite cell surface). The cell and molecular biologic inhibitor type-1 (PAI-1) basis for such a tightly regulated process is now beginning to be understood and is reviewed herein. (Pediatr Res 38: 835443,1995) The fundamental processes responsible for growth and de- that the muscle substance which combines with nicotine or velopment in man are exceedingly complex. The availability of curare is not identical with the substance which contracts. It is cell nutrients as well as growth factors and hormones is convenient to have a term for the specially excitable constitu- essential for normal tissue differentiation. One of the major ent, and I have called it the receptive substance. It receives the mechanisms responsible for delivery of these nutrient mole- stimulus, and by transmitting it, causes contraction" (1). These cules/growth factors to cells is via the process termed RME. two principles-the recognition capacity for specific ligands This system is responsible for the bulk of macromolecular and the subsequent ability of the ligand-receptor complex to transport into the developing egg, across the placenta, across initiate a biologic response-form the basis of our current the neonatal gut, and into the cells of every organ during understanding of receptor biology. However, the recent explo- human development. In addition to the receptors mediating sive growth in the basic biologic understanding of receptor endocytosis and macromolecular transport, cellular receptors biology in the past few years precludes an exhaustive review of govern transmembrane signaling (e.g. for hormones and neu- all these systems. Thus, instead of a superficial overview of a rotransmitters) as well as intracellular signals (e.g. for steroid multitude of systems, I have concentrated on a focused review and thyroid hormones). of one such process, RME. Receptor biology has a long and glorious history. Curare, RME is a specific cellular biologic process by which various used for centuries by the Indians of the Amazon basin as an macromolecules bind to cell surface receptors and are subse- arrow poison, was brought to England by Sir Walter Raleigh in quently internalized and trafficked within the cell. Ligands the 16th century. Claude Bernard began a systematic investi- internalized via RME represent a wide variety of macromole- gation of its action in 1850. But it was J. N. Langley, exam- cules with varying physiologic activities including: nutrient ining the antagonistic effect of curare on nicotine stimulation of provision [e.g. LDL (2, 3), transferrin (4)]; modified molecules skeletal muscle nearly a century ago, who concluded: "Since from the circulation [e.g. ASGP (5, 6), plasminogen activator- neither curare nor nicotine, even in large doses, prevents direct inhibitor complexes (7)]; hormones [e.g. insulin, EGF (8, 9)]; stimulation of muscle from causing contraction, it is obvious and some lysosomal enzymes (10). In addition, certain viruses and toxins (1 1, 12) use this pathway to gain entrance to the cell. Manuscript dedicated to Professor H.K.A. Visser in honor of his retirement. This review provides a general description of the cellular Correspondence and reprint requests: Dr. Alan L. Schwartz, Department of Pediatrics mechanisms and physiology of and intracellular traffick- (Box 8116), Washington University School of Medicine, 660 South Euclid Ave., St. RME Louis, MO 63 1 10. ing of ligand-receptor complexes, the specific receptors com- 836 SCHW monly found on liver cells, the signals responsible for endo- that membrane lipids flow toward coated pit areas (20), sug- cytosis and intracellular targeting and the regulation of the gesting that all membrane proteins may be carried passively to RME pathway. coated pits (21, 22). However, only those receptor proteins mediating endocytosis are "trapped" via specific signals in the MECHANISMS OF RME cytoplasmic tail which interact with structural components of Most cellular functions require an intracellular environment the coated pit (see below). isolated from that of the extracellular space. The cell mem- Coated pits. The coated pit is the cellular machinery for brane provides such a physical barrier to control macromole- ligand-receptor internalization. The name derives from the cule movement. RME provides one major pathway for the characteristic fuzzy, basket-like coat on the cytoplasmic sur- trafficking of extracellular molecules into the cell and involves face of the cell membrane as observed with electron micros- the binding of a ligand to a specific cell surface receptor, copy (23). In most cells coated pits occupy approximately 2% clustering of the ligand-receptor complexes in coated pits, of the plasma surface area (24, 25). Coated pits continuously invagination and pinching off of the coated pits to form coated invaginate and transform into coated vesicles at a rate of vesicles, and delivery of coated vesicles to discrete membrane- approximately 3 X lo3 min-'cell-' (26). This continuous limited cytoplasmic organelles (e.g. endosome1CURL). A transformation requires rapid replenishment of cell surface model of RME is depicted in Figure 1. Each step is discussed coated pits probably via coated pit recycling (27). Although the in detail below. number of coated pits at the cell surface may be regulated Receptor movement to coatedpits. Entry of ligand-receptor under certain conditions (28), in general this step of RME complexes into cells occurs through specialized membrane appears to be constitutive. areas called coated pits. Clustering of receptor proteins within The coat of both coated pits and coated vesicles consists of coated pits is the initial step of RME. This process may be a highly ordered array of specific macromolecules organized either spontaneous or triggered by ligands. Some receptors, into a polygonal lattice (29). Studies on these components such as those for LDL (13) and transferrin (14), appear to be revealed that the basic structural unit of the coat is composed clustered in coated pits independent of prior ligand binding. of three molecules of clathrin heavy chain (180 kD) and three Other receptors, including those for EGF (15), insulin (16), and molecules of clathrin light chain (33-36 kD). The three light ASGP (17), are evenly distributed over the plasma membrane. chains overlap with the three heavy chains to give rise to a Several studies suggest that the rate of receptor diffusion on the three-legged structure termed "triskelion" (30, 3 1). In addition cell surface plasma membrane is sufficient to explain the to clathrin heavy and light chains, coated vesicles also contain movement into coated pits (18, 19). Other evidence suggests a second major class of proteins which have been termed adaptor proteins or AP (32-35). Two heterotetrameric, struc- turally related classes of AP are present in most cells. AP1 consists of 100-1 10 kD, 7,and P' AP and two smaller subunits L of 47 and 49 kD. AP2 consists of 100-1 10 kD a and P AP and GOLGl TGR two smaller subunits of about 50 and 17 kD (35, 36). AP appear as barrel-shaped molecules with two appendages at- tached via a flexible stalk (37). AP appear to function in clathrin assembly. The positioning of AP between the clathrin lattice and the vesicle membrane suggests that AP function in mediating clathrin binding to vesicle membranes. Recent stud- ies suggest that the P or P' subunits mediate the binding to clathrin (38), whereas the other subunits may function in binding receptor molecules (29). The morphology of coated pits has been observed in replicas of cells that are quick-frozen, freeze-fractured, and deep-etched (39). The coats observed under these conditions appeared to form by piecemeal assembly, not by coalescence of large LYSOSOME patches. Some typical lattices seen on the inner surfaces of chick fibroblasts are shown in Figure 2, which is arranged to Figure 1. RME. Ligand molecules bind to specific cell surface receptors at illustrate a progressive increase in the extent of microcage the plasma membrane. After clustering in coated pits and internalization via nucleation around the edges of lattice. Although the precise coated vesicles, the ligand-receptor complexes are delivered to the endosomal sorting compartments in the peripheral cytoplasm. Within these tubulovesicu- molecular mechanisms of coated