CHEMICAL BIOLOGY

Receptor-Mediated Endocytosis

Blake R. Peterson, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania

doi: 10.1002/9780470048672.wecb151

Specific receptors on the surface of mammalian cells actively internalize cell-impermeable ligands by the mechanism of receptor-mediated endocytosis (RME). This process is critical for the acquisition of nutrients, signal transduction, development, neurotransmission, and cellular homeostasis. Binding of ligands to internalizing receptors on the plasma membrane results in clustering of the complex in clathrin-coated pits or other dynamic membrane regions. Invagination of these regions yields intracellular vesicles that fuse to form membrane-sealed endosomes. Receptors typically dissociate from ligands in these acidic compartments, which allows the free receptor to cycle back to the cell surface, whereas ligands are often degraded on delivery to lysosomes, which liberates amino acids and other nutrients. By mimicking endogenous ligands, certain protein toxins, viruses, and other pathogens exploit RME to enter the cytoplasm or reach other intracellular destinations. Similarly, artificial delivery systems that mimic ligands or receptors can enhance efficiently the cellular uptake of impermeable molecules, including drugs, proteins, and nucleic acids. Advances in small-molecule probes, structural biology, and genetic methods are beginning to illuminate the complex mechanisms of this process at the molecular level.

The plasma membrane of eukaryotic cells encapsulates the inner cellular machinery, thereby protecting fragile biologic structures from potentially toxic or opportunistic extracellular materials. Only small hydrophobic molecules can penetrate rapidly this lipid bilayer through passive diffusion. More polar essential amino acids, sugars, and ions access the cell interior by interacting with membrane proteins that function as selective pumps or channels. For many other cell-impermeable small molecules, macromolecules, and particles to access the cell interior, cells must facilitate uptake actively, with regions of the plasma membrane functioning to capture solutes by invaginating and pinching off to form intracellular vesicles. This process is termed endocytosis, which represents multiple related mechanisms for the internalization of extracellular molecules (1). Endocytosis is divided into two primary categories: phagocytosis (cell eating) and pinocytosis (cell drinking). Phagocytosis enables the uptake of large particles, including intact bacteria and yeast, through an actin-mediated mechanism that is generally restricted to specific cell types, such as macrophages, monocytes, and neutrophils. Pinocytosis, by contrast, occurs in all nucleated mammalian cells and involves the active invagination of small regions of cellular plasma membranes to capture solutes within vesicles of less than 200 nm in diameter. These vesicles fuse in the cytoplasm to form membrane-sealed compartments termed endosomes, and their contents are sorted to allow trafficking to specific destinations. Pinocytosis can involve the nonspecific uptake of extracellular fluid, as well as the uptake of specific molecules in the extracellular environment, mediated by receptors on the plasma membrane. Some mechanisms of endocytosis operate rapidly and continuously. In cultured fibroblasts, under physiologic conditions, membrane equivalent to the entire cell surface is perpetually internalized with a half-life of 15 to 30 minutes (2). Most pinocytic pathways involve specific interactions of receptors with ligands. In receptor-mediated endocytosis (RME), internalizing receptors on the cell surface bind cell-impermeable ligands to concentrate ligands in the cell. This mechanism is thousands of times more efficient than nonspecific pinocytosis for the cellular acquisition of nutrients and other impermeable molecules. The receptors involved in RME comprise a structurally diverse group of biomolecules that project ligand-binding motifs into the extracellular environment. Cell-impermeable small molecules, lipids, peptides, proteins, nucleic acids, and carbohydrates are internalized by RME, which enables the consumption of nutrients, elimination of pathogens, and termination of signals initiated by extracellular stimuli. RME followed by subsequent exocytosis of the ligand from one side of the cell to another is termed transcytosis, and this mechanism allows the delivery of nutrients across membrane barriers, such as the blood-brain barrier (3). By exploiting RME, certain viruses, protein toxins, and other pathogens invade cells and cause disease (4). However, to our benefit, drug and other delivery systems that mimic properties of ligands or receptors can be used to access these natural pathways across biologic membranes (5-8).

Internalizing Cell-Surface Receptors and Their Ligands

Cell-surface receptors involved in RME range from macromolecular proteins, which span the plasma membrane, to small glycolipids, which are anchored only to the plasma membrane outer leaflet. Structural representations of several receptors and ligands involved in this process are shown in Fig. 1. X-ray crystal structures of the extracellular domains of the low density lipoprotein (LDL) receptor (9), the transferrin receptor (TFR) (10), the human growth hormone receptor (11), the bovine rhodopsin (12), and the FcyRIIIB (CD16) (13) are shown as part of a composite image that illustrates the nature of attachment of the receptor to the plasma membrane. The small glycolipid receptor ganglioside GM1 is shown to the right, rendered as a molecular model (14). Structures of cognate ligands are positioned above or as a complex with receptor extracellular fragments. These ligands include the structure of LDL determined by cryoelectron microscopy (shown reduced in scale compared with the receptor) (15), transferrin, human growth hormone, the Fc fragment of human IgG, and cholera toxin (16). Brief descriptions of these and related receptors and ligands are provided in the following sections. Other representative examples of receptors and ligands involved in RME are listed in Table 1.

The LDL receptor: a macromolecular membrane-spanning protein critical for cellular uptake of cholesterol

Uptake of cholesterol-laden LDL particles by the LDL receptor (LDLR, Fig. 1) is one of the best-characterized examples of RME (9, 17-18-19). The mature LDL receptor is a single pass transmembrane glycoprotein of 839 amino acids (~115KDa, Fig. 1). LDL ligands are characterized as particles of ~22 nm in diameter (~2500 KDa) that comprise a core of ~1500 molecules of cholesterol esters, esterified primarily by linoleic acid, encapsulated by a monolayer of free cholesterol, phospholipids, triglycerides, and a single large protein termed apolipoprotein B-100 (apo-B, ~550KDa). By recognizing the protein component of LDL, the LDLR enables cells in all tissues of vertebrate animals to internalize exogenous cholesterol, which is a key building block required for the biosynthesis of steroid hormones, bile acids, and cellular membranes. By interacting with the protein clathrin, which forms coated pits on the cytosolic face of the plasma membrane, the LDLR constitutively delivers LDL into endosomes, followed by cycling of the receptor back to the cell surface. Inherited mutations in the LDLR that disrupt endocytosis, and thereby increase serum LDL, have been shown to accelerate atherosclerosis in patients with familial hypercholesterolemia (17). Rapidly proliferating cells have a particularly high demand for cholesterol because mammalian plasma membranes are composed of one-third protein and two-thirds lipid plus ~30% of the cellular plasma membrane lipids are cholesterol (20). For this reason, the LDL receptor is often overexpressed on cancer cells, and LDL receptors provide a target for the selective delivery of anticancer and tumor imaging agents (21, 22). The LDLR is also a portal exploited by Hepatitis C virus and other Flaviviridae viruses to penetrate into cells (23).

The transferrin receptor: a homodimeric transmembrane protein that enables cellular uptake of iron

Iron is an essential nutrient that functions as an enzyme cofactor in redox reactions and plays a structural role through ligand coordination. Under physiologic conditions, iron can be converted readily between the ferrous (Fe²⁺) and the ferric (Fe³⁺) oxidation states. However, ferrous iron is dangerous to living cells because it can generate hydroxyl radicals that oxidatively damage proteins, nucleic acids, and lipids. Additionally, iron in the ferric (Fe³⁺) oxidation state forms a highly insoluble hydroxide complex that is not readily available to cells. In vertebrate animals, ferric iron is transported in serum bound to the protein transferrin (TF), which is a bilobed glycoprotein of 80 KDa in humans (Fig. 1) (10). This protein binds Fe³⁺ using a synergistic anion, typically carbonate, two Tyr, one His, and one Asp residue. Cellular uptake of TF is mediated by the transferrin receptor (TFR, Fig. 1), which is a homodimeric transmembrane protein of 85 KDa in humans that binds two diferric transferrin ligands. Internalization of TF by RME results in the release of Fe³⁺ in acidic endosomal compartments. However, the apo-TF remains bound to TFR, the receptor-ligand complex cycles back to the plasma membrane, and apo-TF is released from the TFR at neutral pH. Ferric iron is reduced to the ferrous state within endosomes, and the iron transporter DMT1 delivers the Fe²⁺ ion into the cytoplasm. Like the LDLR, the TFR is upregulated on certain cancer cell lines, and drugs conjugated to transferrin have been used as targeted delivery systems (5). In mice, the mouse mammary tumor virus exploits the TFR to enter cells (24).

Figure 1. Representative structures of receptors and ligands involved in receptor-mediated endocytosis. The gray bar at the bottom of the figure represents the cellular plasma membrane. From left to right, X-ray crystal structures of the extracellular domains of the human LDL receptor, the human transferrin receptor, the human growth hormone receptor, bovine rhodopsin, and FcyRNIB are shown illustrating the nature of attachment to the plasma membrane. A molecular model of the glycolipid ganglioside GM1 is on the far right. A structure of LDL determined by electron cryomicroscopy (27 A resolution) is shown on the upper left (not drawn to scale; image courtesy of Dr. Wah Chiu, Baylor College of Medicine). Other ligands shown from left to right include receptor-bound transferrin, receptor-bound human growth hormone, receptor-bound Fc region of human IgG, and the B-subunit of cholera toxin.

Receptors for Growth Factors and Hormones

Human growth hormone (GH1, Fig. 1), epidermal growth factor (EGF), insulin (INS), platelet-derived growth factor (PDGF), and many cytokines bind receptors that activate intracellular tyrosine kinase activity. The major isoform of GH1 is a protein of 191 amino acids (22 KDa) that functions in part to stimulate the growth of bone and internal organs in children. As shown in Fig. 1, the human growth hormone receptor (GHR), which is a member of the cytokine-hematopoietin receptor superfamily, comprises a transmembrane glycoprotein of 620 amino acids (130 KDa) (25). Binding of GH1 results in dimerization and conformational changes in the GHR that initiate cellular signaling via recruitment and activation of tyrosine kinases. The GHR is internalized constitutively via clathrin-coated pits, and both this receptor and its ligand are degraded by proteolysis in lysosomes, which provides a mechanism to terminate the extracellular signal. The receptors for EGF and insulin are receptor tyrosine kinases (RTKs) that become internalized only upon binding of ligands. The EGFR family of RTKs includes EGFR (HER1, erbB-1), HER2 (erbB-2), HER3 (erbB-3), and HER4 (erbB-4). Upregulation of expression or the production of activating mutants of this family is known to cause several cancers (26). By binding its extracellular domain, the FDA-approved monoclonal antibody drug Herceptin downmodulates HER2, thereby inhibiting the proliferation of the subset of breast cancers that overexpress this receptor.

G-protein-coupled receptors

G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMs), are the largest known superfamily of proteins. They are involved in all types of responses to stimuli, from intercellular communication to the senses of vision, taste, and smell. They respond to diverse ligands ranging from photons (e.g., rhodopsin, Fig. 1) to small molecules (e.g., binding of epinephrine to the β2-adrenergic receptor) and proteins (e.g., chemokine receptors). Binding of ligands to the extracellular or transmembrane domains of these proteins causes conformational changes that relay a signal to intracellular G proteins that trigger additional cellular responses. Many GPCRs undergo RME by binding to intracellular arrestin proteins that associate with clathrin. The importance of GPCRs in normal biologic processes and disease has made this family of proteins the target of up to 50% of all modern drugs.

Receptors anchored to the plasma membrane by lipids: Glycosylphosphatidylinositol (GPI)-anchored proteins and glycolipids

Some cell-surface receptors are attached to the plasma membrane by lipids that penetrate only into the outer leaflet of the bilayer. Posttranslational modification of proteins with GPI-lipids allows proteins such as folate receptor-2 (FOLR2) to attach to the cell surface and promote RME of the vitamin 5-methyltetrahydrofolate. Folate receptors are upregulated in certain cancers, and folate derivatives have been linked to drugs and molecular probes to treat and image certain tumors. The related GPI-linked receptor FcyRIIIB (CD16, 26.2KDa, Fig. 1) is involved in the immune response. This receptor binds the invariant Fc region of immunoglobulin-G to promote RME of this ligand. Much smaller glycolipids also participate in RME. Gan- glioside GM1 (Fig. 1), a 1.6 KDa glycolipid, enables the protein cholera toxin (16) and the nonenveloped virus SV40 to penetrate into cells upon binding to its pentasaccharide headgroup (27).

Because of the lack of a direct connection to clathrin via a cytoplasmic region, the endocytosis of GPI-linked proteins and other lipid-linked receptors is slower than the uptake of most transmembrane proteins. Instead of clathrin-mediated endocytosis, the internalization of many lipid-linked receptors has been proposed to involve distinct membrane subdomains termed lipid rafts (28). These domains are enriched in cholesterol and sphingolipids and in some cell types include flask-shaped invaginations termed caveolae (29, 30). Many proteins covalently or noncovalently associated with cholesterol, sphingolipids, or saturated lipids are thought to associate with lipid rafts that segregate and concentrate membrane proteins, regulate signal transduction pathways (31), and control the endocytosis of specific receptors (32). Protein toxins and viruses often exploit receptor-mediated endocytosis involving lipid rafts or clathrin to penetrate into the cell interior (4).

Table 1

Examples of receptors and ligands involved in RME. Specific gene symbols of representative human receptor and ligand proteins are listed in parentheses. TM: transmembrane protein; GPI: glycosylphosphatidylinositol anchored protein; 7TM: Seven-transmembrane protein.

Receptor-Mediated Endocytosis Visualized by Confocal Laser Scanning Microscopy

Microscopy has been used extensively to investigate mechanisms of RME. Electron microscopy was instrumental in the identification of clathrin-coated pits, endosomes, and other cellular features involved in this process (33). More recently, confocal laser scanning microscopy of living cells has been employed to investigate the uptake of fluorescent ligands, the influence of molecular probes, and the localization of fluorescent receptors and other proteins during endocytosis. An example of RME as imaged by confocal microscopy is shown in Fig. 2. In this figure, Jurkat lymphocyes, a human helper-T cell line, was treated with a mixture of transferrin conjugated to the bright green fluorophore AlexaFluor-488 and cholera toxin B-subunit conjugated to the red fluorophore AlexaFluor-594. After treatment for 5 minutes, transferrin is internalized by its receptor substantially more rapidly than cholera toxin, which remains partially localized at the cellular plasma membrane. Uptake of these proteins results in delivery in part into distinct early endosomal compartments, which is consistent with significant differences in the mechanisms of endocytic uptake. After 1 hour of treatment, fluorescent transferrin can be localized in early endosomes and the endosomal-recycling compartment, whereas the cholera toxin B-subunit distinctly traffics to the trans-golgi network of living cells. In cells treated with holo-cholera toxin comprising the B-subunit and the catalytic A-subunit, the toxin would traffic further to the endoplasmic reticulum, which would enable the release of the toxic A-subunit into the cytoplasm.

Figure 2. Confocal laser scanning (left panels) and differential interference contrast (DIC, right panels) micrographs of Jurkat lymphocytes treated with green fluorescent transferrin (610 nM) and red fluorescent cholera toxin B-subunit (160 nM). Cells in panel A were treated for 5 minutes, and cells in panel B were treated for 1 hour with the fluorescent protein conjugates at 37° C. The green fluorescence of the transferrin-AlexaFluor488 conjugate is shown in the upper left. The red fluorescence of cholera toxin-AlexaFluor 594 conjugate is shown in the lower left. Colocalization is shown in yellow and overlaid on the DIC image in the lower right. Scale bar = 10 μm. Micrographs courtesy of Ms. Sutang Cai, Penn State University.

Mechanisms of Receptor-Mediated Endocytosis

Cell-surface receptors are involved in both phagocytosis and pinocytosis. At least four distinct mechanisms of pinocytosis have been characterized: macropinocytosis, clathrin-mediated endocytosis, raft/caveolae-mediated endocytosis, and clathrin- and caveolae-independent endocytosis (1). Selected receptor- mediated aspects of these mechanisms are outlined below.

Phagocytosis

Phagocytosis is an actin-mediated mechanism of RME predominantly employed by specialized cells such as macrophages, neutrophils, and monocytes (34). This process allows these cells to clear large pathogens, such as bacteria and yeast, or debris, such as the remnants of dead cells, or deposits of cholesterol and other lipids in arteries. Binding of specific cell-surface receptors to their ligands triggers phagocytosis. For example, Fc receptors on macrophages bind the Fc region of antibodies that coat surface antigens on pathogens or particles. These recognition events activate signaling cascades involving Rho-family GT-Pases that trigger the assembly of the cytoskeletal protein actin and promote the extension of membrane segments that engulf the antibody-coated target. Dynamin, a large GTPase involved in the scission of vesicles from membranes, seems to be critical for phagocytosis (35). Ligands of receptors that trigger phagocytosis include phosphatidylserine exposed on apoptotic cells resulting from inflammation, tissue damage, or development. Other ligands include lipopolysaccaride, mannose residues, fucose residues, complement proteins, and fibronectin.

Macropinocytosis

Macropinocytosis is another actin-mediated uptake mechanism that can be induced transiently in most mammalian cells. Binding of growth factors, such as platelet-derived growth factor (PDGF) and other signals to receptors, activates signaling cascades involving Rho-family GTPases, which triggers the actin-driven formation of membrane protrusions. The formation and collapse of these plasma membrane ruffles generates large endocytic vesicles, termed macropinosomes, with diameters of 0.5 to 2.5 pm. Constitutive macropinocytosis by dendritic cells allows the efficient capture of exogenous antigens; presentation on the cell surface bound to MHC molecules provides a mechanism for stimulation of immune responses (36). Macropinocytosis also may be involved in the downregulation of activated signaling molecules.

Clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) is responsible for the uptake of about 50% of all ligands internalized by cell-surface receptors. This process is critical for the continuous uptake of nutrients, intercellular communication during development, and regulation of signal transduction throughout life. CME modulates cellular signaling by controlling the levels of cell-surface receptors and downregulating activated signaling receptors. CME affects cell and serum homeostasis by controlling the internalization of membrane pumps involved in the transport of ions and small molecules across the plasma membrane. In neurons, CME promotes the uptake of voltage-gated ion channels, which affects the strength of synaptic transmission, and it is involved in the recycling of membrane proteins of synaptic vesicles after neurotransmission.

Figure 3. Pathways of endocytic trafficking of receptors and ligands. The model illustrates uptake of LDL mediated by the LDLR, diferric transferrin (TF-Fe³⁺) internalized by the transferrin receptor (TFR), and entry of cholera toxin (CTX) and simian virus-40 (SV40) after binding to ganglioside GM1. Related trafficking of glycosylphosphatidylinositol-anchored proteins (GPI-AP) is also shown. The LDLR and TFR concentrate in clathrin-coated pits and initially deliver ligands into endocytic vesicles that fuse with sorting endosomes. The acidic environment of sorting endosomes dissociates most receptor-ligand complexes, and membrane proteins typically exit these compartments rapidly and return directly to the plasma membrane or are shuttled to the endocytic recycling compartment (ERC or recycling endosome). LDLRs and TFRs are recycled from the ERC back to the cell surface. LDL is sorted to late endosomes and lysosomes, where it is degraded and releases cholesterol and amino acids into the cell. TF-Fe³⁺ releases iron in the acidic sorting endosome, but under acidic conditions, iron-free TF remains bound to the TFR. Upon return to the cell surface, at neutral pH, iron-free TF dissociates from the receptor. Binding of CTX and SV40 to GM1 primarily results in endocytic uptake via uncoated pits and caveolae, respectively. Internalized GPI-APs, CTX, and SV40 traffic through either the GPI-anchored protein-enriched early endosomal compartment (GEEC) or a related compartment termed the caveosome. SV40 moves from the caveosome directly into the endoplasmic reticulum (ER). In contrast, CTX exits the GEEC and passes through the trans-golgi network to the ER. From the ER, CTX and SV40 penetrate into the cytosol, resulting in toxicity or infection, respectively. The t_1/2 values shown are approximate and are cell-type dependent.

The protein clathrin comprises 190-KDa and 25-KDa subunits that form a basket-like structure on the cytoplasmic face of the plasma membrane. These subunits assemble as complexes with adaptor proteins into highly ordered polygonal arrays that define pits on the cell surface. When clathrin-coated pits invaginate and pinch off, they form clathrin-coated vesicles (CCVs) with a diameter of ~120nm. Assembly of a CCV in cultured cells takes ~1 minute, and hundreds to a thousand or more can form every minute. Clathrin-mediated endocytosis is the best-characterized mechanism of ligand uptake, and interactions of clathrin with receptors such as LDLR and TFR result in clustering of receptor-ligand complexes in clathrin-coated pits (Fig. 3). The LDLR and TFR interact with clathrin via adapter proteins such as the autosomal recessive hypercholesterolemia (ARH) protein and AP-2, respectively, but the LDLR also interacts directly with clathrin (37). GPCRs are linked to clathrin via an adapter-like protein, β-arrestin, which interacts with AP-2. Specific peptide sequences bind adapter proteins and couple receptors to the clathrin-controlled endocytic machinery. The best-defined coated pit internalization signals are the tyrosine-based FxNPxY (F = phenylalanine, x = any amino acid, N = asparagine, P = proline, and Y = tyrosine) motif found in the LDLR, the YxΨθ (Y = tyrosine, x = any amino acid, Ψ = a bulky hydrophilic amino acid, and θ = a hydrophobic amino acid) motif of the TFR, and a dileucine motif of the insulin and β₂-adrenergic receptors (1). Internalization of LDL and transferrin is a constitutive and rapid clathrin-mediated process. However, binding of other ligands, such as EGF to EGFR or epinephrine to the β₂-adrenergic receptor, induces internalization via clathrin (32). Expression of dominant negative mutants of dynamin, the AP-2 binding partner Eps15, and its binding partner epsin have been used to inhibit CME and to identify mechanisms controlling receptor-mediated endocytosis.

Receptor-ligand complexes clustered in clathrin-coated pits are internalized when the plasma membrane invaginates, GTP- driven conformational changes of dynamin trigger membrane scission by forming a helix around the neck of a nascent vesicle (35), and the endocytic vesicle undocks from the membrane. The pH of these internalized vesicles drops as a consequence of the activation of proton pumps, which occurs in conjunction with the opening of chloride channels. Fusion of these vesicles in the cytoplasm yields larger acidic (pH ~ 6) sorting endosomes, relatively short-lived compartments that accept incoming material for only about 5 to 10 minutes (2). The decrease in pH and the intrinsic tubular-vesicular geometry of sorting endosomes, defined by a high membrane surface area and low solute volume, facilitates the dissociation of the LDL receptor from LDL in these compartments. In contrast, the protein component of TF remains associated with the TFR in endosomes, but the TF releases its bound ferric iron under these conditions. Both the free LDLR and the iron-free TF-TFR subsequently traffic to the endocytic recycling compartment (ERC), a long-lived organelle, before cycling back to the cell surface. In this way, the LDL receptor can be reused up to several hundred times during its ~ 20-hour lifespan. Upon return of the TFR to the plasma membrane and exposure to neutral pH, the iron-free TF protein dissociates from the receptor, which enables another round of uptake of iron-loaded ligand. Sorting endosomes that contain free LDL mature to form more acidic late endosomes (pH ~ 5.5), and these compartments subsequently fuse with lysosomes, more acidic organelles (pH ~ 5) that contain hydrolytic enzymes. Hydrolysis of cholesteryl esters, protein, and other components of LDL in lysosomes liberates these nutrients for use by the cell (18, 38). For some receptors, such as the EGFR and GPCRs, ubiquitination of receptor lysine residues is a signal for targeting to lysosomes, which provides a mechanism for receptor downregulation by endocytosis.

Receptor-mediated endocytosis via caveolae and other mechanisms

Lipid raft microdomains of mammalian plasma membranes are thought to regulate the endocytosis of specific ligands (32, 39). These domains are envisaged to comprise islands of ordered cholesterol, sphingolipids, and saturated lipids that move within the plane of disordered unsaturated lipids. The formation of lipid rafts depends on the availability of cholesterol in the membrane, and agents that sequester cholesterol, such as P-cyclodextrins, can selectively disrupt these microdomains. Dominant negative mutants of dynamin also block endocytosis via this mechanism. GPI-anchored receptors, such as FcyRIIIB and folate receptors, and glycolipids, such as ganglioside GM1, are thought to reside in lipid rafts and become internalized at least partially by caveolae-mediated and clathrin-independent/caveolin-independent endocytic pathways.

Caveolae represent a subset of lipid rafts found on specific cell types, including adipocytes, endothelia, and muscle cells. These lipid rafts can be observed by electron microscopy as distinctive flask-shaped pits of ~60 nm in diameter and include proteins of the caveolin family on the cytoplasmic face of the pit. Lymphocytes and many neuronal cells do not express caveolins and lack these morphological membrane features. Although caveolae are considered relatively static structures on the cell surface, they can become internalized on binding of ligands to receptors that associate with these raft subdomains. After activation, caveolae are internalized with relatively slow kinetics (half-life > 20 min) compared with CME (1).

In cells bearing caveolae, binding of simian virus-40 (SV40) to ganglioside GM1 in these subdomains triggers internalization into compartments termed caveosomes. This DNA virus subsequently undergoes trafficking to the endoplasmic reticulum, which is a destination that allows escape into the cytoplasm through an unknown mechanism, before the virus enters the nucleus. In cell lines lacking caveolae, other lipid raft domains are thought to promote delivery into distinct compartments termed GPI-anchored protein early endosomal compartments (GEECs) (37). For example, binding of cholera toxin to ganglioside GM1 results, at least in part, in uptake through uncoated pits and in trafficking to the GEEC through the trans-golgi network eventually to the ER, where a catalytically active fragment can escape into the cytosol and exert toxic effects. Cholera toxin, however, is not a specific marker for raft-mediated endocytosis; this protein is known to be internalized by three distinct mechanisms: clathrin coated pits, caveolae, and a clathrin- and caveolin-independent pathway (40). The mechanism that couples recognition of the glycolipid on the outer leaflet of the membrane to the clathrin machinery on the inner leaflet is unknown. The relationship between lipid rafts and clathrin is also not yet well defined. For example, the EGFR is internalized through a mechanism that seems to simultaneously involve both lipid rafts and clathrin (41). Mechanisms of endocytosis that are independent of both clathrin and caveolin are not well understood.

Molecular and Cellular Probes of Receptor-Mediated Endocytosis

Small molecules, proteins, and genetic constructs that activate, block, or label specific endocytic components or pathways represent key tools for studies of RME. Representative examples of probes of RME and related cellular processes are provided in Table 2. An overview of these approaches is provided in the following subsections.

Small-molecule regulators of phagocytosis and macropinocytosis

Many compounds that perturb the cellular cytoskeleton affect phagocytosis and macropinocytosis. Binding to actin filaments by the natural product cytochalasin D blocks both of these uptake mechanisms. Disruption of microtubules by the antimitotic agents colchicine and nocodazole inhibits macropinocytosis and affects some mechanisms of phagocytosis. The diuretic drug amiloride, which is an inhibitor of Na⁺/H⁺ antiporters, selectively blocks macropinocytosis. By activating protein kinase C, phorbol esters represent a class of small molecules that promote macropinocytosis.

Table 2

Examples of small molecules, altered cell culture conditions, proteins, and genetic constructs used to probe RME. CID: chemical inducer of dimerization.

Small molecules and modified cell culture conditions that block CME

Clathrin-mediated endocytosis can be blocked by several pharmacologic inhibitors, including the antipsychotic drug chlorpromazine (Thorazine), the natural product ikarugamycin, and the antiviral drug amantadine. The metabolic poisons phenylarsine oxide and sodium azide also block CME but additionally inhibit protein synthesis. Culture of cells under conditions that deplete potassium or calcium, treatment of cells with hypertonic sucrose, or acidification of the cytoplasm by addition of acetic acid to media, have also been used to block this mechanism of cellular uptake. However, many of these treatments tend to be relatively nonspecific and inhibit multiple cellular uptake processes. To block selectively dynamin-dependent endocytic mechanisms, a small molecule termed dynasore has been identified as a specific inhibitor of dynamin (42). Another strategy for inhibiting CME with high specificity uses a chemical inducer of dimerization (CID) combined with a genetic approach to promote aberrant oligomerization of clathrin in cells transfected with a clathrin fusion protein. This system can rapidly and reversibly inhibit 70% of the endocytosis of TFR (43).

Inhibitors of raft/caveolin-mediated endocytosis

Lipid raft domains of plasma membranes are enriched in cholesterol and sphingolipids. As a consequence, compounds that extract or sequester cholesterol, such as β-cyclodextrins, nystatin, and filipin, can block selectively endocytosis of cholera toxin, GPI-linked proteins, and other receptors that associate with lipid rafts and caveolae. However, cholesterol is also critical for CME, secretion of proteins, and the actin network. Therefore, conditions designed to affect selectively raft-mediated endocytosis by perturbing cholesterol levels must be carefully controlled to avoid disrupting other mechanisms of endocytosis (40).

Small-molecule probes of other endocytic trafficking pathways

Phosphatidyl-inositol-3-OH kinase (PI(3)kinase) plays an important role in the fusion of endosomes. Phosphatidyl-inositol-3-phosphate (PI(3)P), a product of this enzyme, is enriched in early endosomes, and blocking PI(3)kinase activity with the small molecule wortmannin prevents endosome fusion. This fungal natural product has been shown to inhibit the endocytosis of transferrin, horseradish peroxidase, and albumin (44, 45).

Fluorescent probes of endocytosis

Fluorescent small molecules and proteins represent powerful tools for labeling ligands, receptors, and other targets involved in endocytosis. By conjugating ligands of receptors to flu- orophores, the uptake of these molecules can be analyzed by fluorescence microscopy, flow cytometry, and other methods. Small-molecule fluorophores have been installed through site-specific protein labeling on modified cell-surface receptors, such as TFR (46), NK₁ (47), and EGFR (48), which are expressed in transfected cell lines. Studies of trafficking of the TF/TFR complex that combine site-specific protein labeling with analysis by fluorescence resonance energy transfer (FRET) have demonstrated that this method is a powerful tool for studying endocytosis and exocytosis (46).

Green fluorescent protein (GFP) and related fluorescent proteins can be used to label practically any protein or subcellular compartment of living cells (49). Transfection of cells with plasmids that encode appropriately targeted fluorescent fusion proteins has been used to define the plasma membrane, early endosomes, late endosomes, caveolae, the golgi complex, the ER, and other subcellular locations. Several fluorescent small molecules are also available for labeling specific cellular organelles, including endosomes and lysosomes, for analysis by fluorescence microscopy.

Antibody probes and regulators of endocytosis

Immunofluorescence techniques are often used to identify specific cellular targets, including proteins, involved in endocytosis. However, because antibody reagents are not cell permeable, cells typically must be fixed or microinjected to allow binding to intracellular proteins. For example, microinjection of antibodies against clathrin (50) and dynamin (51) has been used to block CME. However, because of their cell impermeability, antibody reagents are limited in studies of dynamic cellular processes (52).

Genetic approaches for targeting specific components of endocytic pathways

Genetic methods, such as the expression of dominant negative proteins and RNA interference (RNAi), represent some of the most highly specific approaches for studies of endocytosis. Dominant negative mutants of clathrin, dynamin, Eps15, and other components of the endocytic machinery have been used widely to probe endocytic pathways. More recently RNAi has emerged as an important new tool for downregulating specific targets, including clathrin, AP-2, and epsin (53). Although these genetic methods have the potential to inactivate proteins in cells with a high degree of specificity, they also have associated limitations. For example, RNAi against clathrin heavy chain and AP-2 can suffer from cross-reactivity with other endocytic pathways (54). Another disadvantage of RNAi is that it can require several days to eliminate the targeted protein, which allows the activation of alternative compensatory mechanisms. The identification of temperature-sensitive alleles that result in inactivation of a specific protein at a defined temperature can also provide powerful tools for studies of dynamic cellular processes such as RME. However, these systems can be difficult to implement in mammalian cells.

Synthetic Mimics of Ligands and Receptors

Mimics of ligands and receptors have been used to promote the endocytic uptake of drugs, proteins, DNA, and other cell-impermeable molecules. Ligands linked to cargo of interest can often access efficiently the cell interior via binding to internalizing cell-surface receptors. Modified natural cell-surface receptors and small synthetic mimics of receptors have been shown to promote the uptake of specific ligands via RME. Examples of mimics of ligands and receptors are shown in Fig. 4.

Figure 4. Examples of mimics of ligands and receptors used to deliver cargo into cells. (a,b) Ligands such as folate can be linked to macromolecules (e.g., nucleic acids and proteins) and small molecules (e.g., drugs, radiochemicals, and fluorophores) to promote cellular uptake. (c) Peptides and small molecules linked to N-alkyl derivatives of 3β-cholesterylamine or 3β-dihydrocholesterylamine mimic cell-surface receptors by cycling rapidly between the plasma membrane and the intracellular endosomes. The synthetic Fc receptor shown in (c) enables treated mammalian cells to internalize human IgG.

Cellular uptake of cargo conjugated to ligands of internalizing receptors

Numerous ligands of cell-surface receptors have been linked to cell-impermeable macromolecules, drugs, and other cargo for delivery into mammalian cells. Macromolecular ligands modified in this way include transferrin (5), LDL (22), growth factors, and antibodies that bind the extracellular domains of cell-surface receptors. An example of an FDA-approved drug that functions in this way is mylotarg, which is a monoclonal antibody drug that comprises the anticancer agent calicheamicin linked to a humanized antibody that binds the CD33 antigen on myeloid leukemia cells (55). Binding of mylotarg to CD33 results in endocytosis of the antibody drug conjugate, release of calicheamicin in endosomes, escape of the drug into the cell nucleus, and antiproliferative effects against targeted cells.

The vitamins folate (vitamin B9) (56, 57) and cobalmin (vitamin B12) (58) have been investigated as vehicles for delivery of impermeable molecules into cells. By binding with high affinity (Kd ~ 10^-10 M) to folate receptors, folate-linked drugs, radiopharmaceuticals, nucleic acids, and nanoparticles can become internalized by RME. Similarly, transcobalamin receptors bind and promote the endocytosis of cobalamin and cobalamin conjugates complexed to soluble transcobalamin (59). This approach has been used to target selectively tumor cells that overexpress these receptors.

Artificial cell-surface receptors

Synthetic mimics of cell-surface receptors have been constructed using plasma membrane anchors derived from N-alkyl-3β-cholesterylamine and related compounds (14). Synthetic receptors comprising protein and other binding motifs linked to this membrane anchor become incorporated rapidly into plasma membranes of mammalian cells. By constitutively cycling between the plasma membrane and the endosomes, cells treated with these compounds gain the capacity to endocytose cell-impermeable ligands. For example, the synthetic Fc receptor shown in Fig. 4 enables human cells that lack Fc receptors to internalize human IgG (60). This strategy, termed synthetic receptor targeting, seems to mimic the initial steps of uptake of cholera toxin mediated by ganglioside GM1. This approach may have applications as a method for drug delivery (61).

A related strategy, termed cellular painting, has been used to incorporate proteins linked to GPI lipid anchors into cellular plasma membranes (62, 63). This approach been used to study cellular signaling, plasma membrane organization, and immunologic responses to modified cell surfaces. Because GPI-linked proteins undergo raft-mediated endocytosis, Jurkat lymphocytes treated with a GPI-linked variant of the immunoglobulin Fc receptor FcyRIII will endocytose ligands that bind this receptor (64). Single-chain antibodies covalently linked to lipids have also been used to construct related cell-surface receptors (65).

Metabolic cell-surface engineering to promote the endocytic uptake of ligands

Metabolic cell-surface engineering has been used to modify carbohydrate components of cell-surface receptors and to enable endocytic uptake of impermeable ligands (66). By feeding cells unnatural sugars, cellular metabolism can be harnessed to display bioorthogonal functional groups, such as ketones and azides from glycoproteins and glycolipids, on cell surfaces. The reaction of these ketones with hydrazine derivatives to yield hydrazones (67), as well as the reaction of azides with modified phosphines in the Staudinger ligation (68), can immobilize molecules on the cell surface and promote delivery of proteins such as the toxin ricin (67).

Future Research Directions

Molecular and cellular probes of RME

The molecular mechanisms controlling CME are beginning to be characterized. However, other mechanisms of RME, particularly those involving lipid rafts, caveolae, and other pathways, are less well understood. Although genetic methods such as RNAi and expression of dominant negative proteins represent important tools for inactivating key players in endocytic pathways, the delayed cellular response associated with many of these approaches can allow compensatory gene expression or other mechanisms that can complicate the analysis. Because small molecules can inactivate rapidly defined protein targets, the identification of highly specific inhibitors of proteins involved in RME may provide the best tools for studying the molecular mechanisms of this process. Screens based on “composite synthetic lethality” are particularly promising for the identification of small molecules that target specific membrane trafficking pathways (69).

Mimicry and modifications of ligands

Mimics of ligands that bind cell-surface receptors and undergo RME are becoming an increasingly important class of targeted therapeutics. For example, folic acid derivatives show substantial potential both in vitro and in vivo for targeting tumor cells that overexpress folate receptors (6). The identification of other modified ligands that can achieve selectivity for specific cells or tissues involved in disease is an important research challenge. The construction of improved linkers between drugs and ligands that allow efficient release of cargo in cells or tissues with temporal control is an area that would benefit from additional research.

Mimicry and modifications of cell-surface receptors

Improvements in site-specific protein labeling methods have great potential for studies of receptor trafficking in living cells (46-48). These methods may overcome some disadvantages of green fluorescent and related fusion proteins, including their high molecular weight and relatively low intrinsic brightness compared with the best small-molecule fluorophores. New methods for site-specific labeling of proteins combined with studies by FRET (46) suggest that this approach could be a powerful tool for tracking the formation of receptor-ligand complexes, endocytosis, and exocytosis in real time.

The construction of artificial cell-surface receptors that function as prosthetic molecules on cell surfaces is another emerging area in chemical biology (14). For drug delivery and related applications, improved methods for disruption of endosomes with low toxicity are needed to release internalized ligands into the cytosol and other subcellular compartments. The construction of artificial cell-surface receptors designed to mimic transmembrane proteins and engage both extracellular ligands and intracellular molecules, such as clathrin or signaling proteins, may also provide interesting new tools for studies of biology.

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Further Reading

Marsh M, McMahon HT. The structural era of endocytosis. Science 1999; 285:215-220.

See Also

Biomacromolecule-Directed (Target-Specific) Drug Delivery: Underlying Factors, Principles and Design

Chemical Events in Neurotransmission;

Endocytosis and Exocytosis, Membrane Dynamics of;

Lipid Domains, Chemistry of

Receptor Tyrosine Kinases

Receptor-Ligand Interactions

Receptors, Chemistry of

Signal Transduction Across Membranes

Virus-Based Drug Delivery