Receptor Mediated Endocytosis and the Sorting of Internalized Proteins

In previous sections we have explored the main pathways whereby secretory and membrane proteins synthesized on the rough ER are delivered to the cell surface or other destinations. Cells also can internalize materials from their surroundings and sort these to particular destinations. A few cell types (e.g., macrophages) can take up whole bacteria and other large particles by phagocytosis, a nonselective actin-mediated process in which extensions of the plasma membrane envelop the ingested material, forming large vesicles called phagosomes (see Figure 5-20). In contrast, all eukary-otic cells continually engage in endocytosis, a process in which a small region of the plasma membrane invaginates to form a membrane-limited vesicle about 0.05-0.1 ^m in diameter. In one form of endocytosis, called pinocytosis, small droplets of extracellular fluid and any material dissolved in it are nonspecifically taken up. Our focus in this section, however, is on receptor-mediated endocytosis in which a specific receptor on the cell surface binds tightly to an extracellular macromolecular ligand that it recognizes; the plasma-membrane region containing the receptor-ligand complex then buds inward and pinches off, becoming a transport vesicle.

Among the common macromolecules that vertebrate cells internalize by receptor-mediated endocytosis are cholesterol-containing particles called low-density lipoprotein (LDL); the iron-binding protein transferrin; many protein hormones (e.g., insulin); and certain glycoproteins. Receptor-mediated endocytosis of such ligands generally occurs via clathrin/AP2-

► EXPERIMENTAL FIGURE 17-27

The initial stages of receptor-mediated endocytosis of low-density lipoprotein (LDL) particles are revealed by electron microscopy. Cultured human fibroblasts were incubated in a medium containing LDL particles covalently linked to the electron-dense, iron-containing protein ferritin; each small iron particle in ferritin is visible as a small dot under the electron microscope. Cells initially were incubated at 4 °C; at this temperature LDL can bind to its receptor but internalization does not occur. After excess LDL not bound to the cells was washed away, the cells were warmed to 37 °C and then prepared for microscopy at periodic intervals. (a) A coated pit, showing the clathrin coat on the inner (cytosolic) surface of the pit, soon after the temperature was raised. (b) A pit containing LDL apparently closing on itself to form a coated vesicle. (c) A coated vesicle containing ferritin-tagged LDL particles. (d) Ferritin-tagged LDL particles in a smooth-surfaced early endosome 6 minutes after internalization began. [Photographs courtesy of R. Anderson. Reprinted by permission from J. Goldstein et al., Nature 279:679. Copyright 1979, Macmillan Journals Limited. See also M. S. Brown and J. Goldstein, 1986, Science 232:34.]

coated pits and vesicles in a process similar to the packaging of lysosomal enzymes by mannose 6-phosphate (M6P) in the trans-Golgi network (see Figure 17-23). As noted earlier, some M6P receptors are found on the cell surface, and these participate in the receptor-mediated endocytosis of lysosomal enzymes that are secreted. In general, transmembrane receptor proteins that function in the uptake of extracellular ligands are internalized from the cell surface during endocytosis and are then sorted and recycled back to the cell surface, much like the recycling of M6P receptors to the plasma membrane and trans-Golgi. The rate at which a lig-and is internalized is limited by the amount of its corresponding receptor on the cell surface.

Clathrin/AP2 pits make up about 2 percent of the surface of cells such as hepatocytes and fibroblasts. Many internalized ligands have been observed in these pits and vesicles, which are thought to function as intermediates in the endo-cytosis of most (though not all) ligands bound to cell-surface receptors (Figure 17-27). Some receptors are clustered over clathrin-coated pits even in the absence of ligand. Other receptors diffuse freely in the plane of the plasma membrane but undergo a conformational change when binding to lig-and, so that when the receptor-ligand complex diffuses into a clathrin-coated pit, it is retained there. Two or more types of receptor-bound ligands, such as LDL and transferrin, can be seen in the same coated pit or vesicle.

Clathrin-coated pit (c)
Transferrin Receptor Membrane

Receptors for Low-Density Lipoprotein and Other Ligands Contain Sorting Signals That Target Them for Endocytosis

As will be discussed in detail in the next chapter, low-density lipoprotein (LDL) is one of several complexes that carry cholesterol through the bloodstream (see Figure 17-28). A LDL particle, a sphere 20-25 nm in diameter, has an outer phospholipid shell containing a single molecule of a large

▲ FIGURE 17-28 Endocytic pathway for internalizing low-density lipoprotein (LDL). Step 1: Cell-surface LDL receptors bind to an apoB protein embedded in the phospholipid outer layer of LDL particles. Interaction between the NPXY sorting signal in the cytosolic tail of the LDL receptor and the AP2 complex incorporates the receptor-ligand complex into forming endocytic vesicles. Step 2: Clathrin-coated pits (or buds) containing receptor-LDL complexes are pinched off by the same dynamin-mediated mechanism used to form clathrin/AP1 vesicles on the trans-Golgi network (see Figure 17-20). Step 3: After the vesicle coat is shed, the uncoated endocytic vesicle (early protein known as apoB-100; the core of a particle is packed with cholesterol in the form of cholesteryl esters (see Figure 18-12). Most mammalian cells produce cell-surface receptors that specifically bind to apoB-100 and internalize LDL particles by receptor-mediated endocytosis. After endocytosis, the LDL particles are transported to lysosomes via the endocytic pathway and then are degraded by lysosomal hydrolases. LDL receptors, which dissociate from their ligands in the late endosome, recycle to the cell surface.

endosome) fuses with the late endosome. The acidic pH in this compartment causes a conformational change in the LDL receptor that leads to release of the bound LDL particle. Step 4|: The late endosome fuses with the lysosome, and the proteins and lipids of the free LDL particle are broken down to their constituent parts by enzymes in the lysosome. Step 5: The LDL receptor recycles to the cell surface where at the neutral pH of the exterior medium the receptor undergoes a conformational change so that it can bind another LDL particle. [See M. S. Brown and J. L. Goldstein, 1986, Science 232:34, and G. Rudenko et al., 2002, Science 298:2353.]

LDL particle

Phospholipid monolayer ApoB protein

LDL receptor

Coated pit

Clathrin

LDL particle

Phospholipid monolayer ApoB protein

LDL receptor

Coated pit

Clathrin

Amino acids

Cholesterol

Amino acids

Cholesterol

At neutral pH, ligand-binding arm is free to bind another LDL particle

Plasma membrane

At neutral pH, ligand-binding arm is free to bind another LDL particle

Late endosome

Plasma membrane

Late endosome

Studies of the inherited disorder familial hypercholes-terolemia led to discovery of the LDL receptor and the initial understanding of the endocytic pathway. An individual with this disorder produces one of several mutant forms of the LDL receptor, causing impaired endocytosis of LDL and high serum levels of cholesterol (Chapter 18). The major features of the LDL endocytic pathway as currently understood are depicted in Figure 17-28. The LDL receptor is an 839-residue glycoprotein with a single transmembrane segment; it has a short C-terminal cytosolic segment and a long N-terminal exoplasmic segment that contains a ^-propeller domain and a ligand-binding domain. Seven cysteine-rich imperfect repeats form the ligand-binding domain, which interacts with the apoB-100 molecule in a LDL particle.

Mutant receptors from some individuals with familial hypercholesterolemia bind LDL normally, but the LDL-receptor complex cannot be internalized by the cell and is distributed evenly over the cell surface rather than being confined to clathrin/AP2-coated pits. In individuals with this type of defect, plasma-membrane receptors for other ligands are internalized normally, but the mutant LDL receptor apparently is not recruited into coated pits. Analysis of this mutant receptor and other mutant LDL receptors generated experimentally and expressed in fibrob-lasts identified a four-residue motif in the cytosolic segment of the receptor that is crucial for its internalization: Asn-Pro-X-Tyr where X can be any amino acid. This NPXY sorting signal binds to the AP2 complex, linking the clathrin/AP2 coat to the cytosolic segment of the LDL receptor in forming coated pits. A mutation in any of the conserved residues of the NPXY signal will abolish the ability of the LDL receptor to be incorporated into coated pits.

A small number of individuals who exhibit the usual symptoms associated with familial hypercholesterolemia produce normal LDL receptors. In these individuals, the gene encoding the AP2 subunit protein that binds the NPXY sorting signal is defective. As a result, LDL receptors are not incorporated into clathrin/AP2 vesicles and endocytosis of LDL particles is compromised. Analysis of patients with this genetic disorder highlights the importance of adapter proteins in protein trafficking mediated by clathrin vesicles. I

Mutational studies have shown that other cell-surface receptors can be directed into forming clathrin/AP2 pits by a different sorting signal: Tyr-X-X-$, where X can be any amino acid and $ is a bulky hydrophobic amino acid. This YXX$ sorting signal in the cytosolic segment of a receptor protein binds to a specific cleft in the ^2 subunit of the AP2 complex. Because the tyrosine and $ residues mediate this binding, a mutation in either one reduces or abolishes the ability of the receptor to be incorporated into clathrin/AP2-coated pits. Moreover, if influenza HA protein, which is not normally endocytosed, is genetically engineered to contain this four-residue sequence in its cytosolic domain, the mutant HA is internalized. Recall from our earlier discussion that this same sorting signal recruits membrane proteins into clathrin/AP1 vesicles that bud from the trans-Golgi network by binding to the p1 subunit of AP1 (see Table 17-2). All these observations indicate that YXX$ is a widely used signal for sorting membrane proteins to clathrin-coated vesicles.

In some cell-surface proteins, however, other sequences (e.g., Leu-Leu) or covalently linked ubiquitin molecules signal endocytosis. Among the proteins associated with clathrin/ AP2 vesicles, several contain domains that specifically bind to ubiquitin, and it has been hypothesized that these vesicle-associated proteins mediate the selective incorporation of ubiquitinated membrane proteins into endocytic vesicles. As described later, the ubiquitin tag on endocytosed membrane proteins is also recognized at a later stage in the endocytic pathway and plays a role in delivering these proteins into the interior of the lysosome where they are degraded.

The Acidic pH of Late Endosomes Causes Most Receptor-Ligand Complexes to Dissociate

The overall rate of endocytic internalization of the plasma membrane is quite high; cultured fibroblasts regularly internalize 50 percent of their cell-surface proteins and phospho-lipids each hour. Most cell-surface receptors that undergo endocytosis will repeatedly deposit their ligands within the

Ligand in lumen

Receptors in vesicle extensions

Ligand in lumen

Receptors in vesicle extensions

▲ EXPERIMENTAL FIGURE 17-29 Electron microscopy demonstrates that endocytosed receptor-ligand complexes dissociate in late endosomes. Liver cells were perfused with an asialoglycoprotein ligand and then were fixed and sectioned for electron microscopy. The sections were stained with receptor-specific antibodies, tagged with gold particles 8 nm in diameter, to localize the receptor and with asialoglycoprotein-specific antibody, linked to gold particles 5 nm in diameter, to localize the ligand (see Figure 5-51). As seen in this electron micrograph of a late endosome, the ligand (smaller dark grains) is localized in the vesicle lumen and the asialoglycoprotein receptor (larger dark grains) is localized in the tubular extensions budding off from the vesicle. [Courtesy of H. J. Geuze. Copyright 1983, M.I.T See H. J. Geuze et al., 1983, Cell 32:277.]

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