17:  17.9 Receptor-Mediated Endocytosis and the Sorting of Internalized Proteins

The LDL Receptor Binds and Internalizes Cholesterol-Containing Particles

Whether ingested in foodstuffs or synthesized in the liver, cholesterol is insoluble in body fluids and must be transported by a water-soluble carrier. Low-density lipoprotein (LDL) is one of several complexes that carry cholesterol through the bloodstream. An LDL particle is a sphere 20 – 25 nm in diameter (Figure 17-45). Its outer surface is a monolayer membrane of phospholipids and cholesterol, in which one molecule of a very large protein, called apo-B, is embedded. Inside is an extremely nonpolar core of cholesterol, all of which is esterified through the single hydroxyl group of cholesterol to a long-chain fatty acid, mainly linoleic acid. Most mammalian cells produce cell-surface receptors that specifically bind and internalize LDL by receptor-mediated endocytosis (see Figure 17-44). After endocytosis, the LDL particles are transported to lysosomes where lysosomal hydrolases degrade the apo-B protein to amino acids and cleave the cholesterol esters to cholesterol and fatty acids. The cholesterol is incorporated directly into cell membranes or is reesterified and stored as lipid droplets in the cell for later use; the fatty acids are used to make new phospholipids or tri-glycerides. Cholesterol also is converted to steroid hormones in adrenal cortical cells and to bile acids in hepatocytes.

The LDL receptor is a single-chain glycoprotein of 839 amino acids with a long N-terminal exoplasmic domain and short C-terminal cytosolic domain (see Figure 17-21). A sequence of 22 hydrophobic amino acids spans the plasma membrane once, presumably as an α helix. The large exoplasmic domain has an N-terminal segment of about 320 residues that is extremely rich in disulfide-bonded cysteine residues. This segment includes a sevenfold repeat of a sequence of 40 amino acids that contains the LDL-binding site. As explained below, residues in the C-terminal cytosolic domain are involved in trapping the LDL receptor in clathrin-coated pits.

Cytosolic Sequences in Some Cell-Surface Receptors Target Them for Endocytosis

As we learn in Chapter 20, cells possess many different cell-surface receptors with binding sites for specific ligands. Only some of these receptors, however, form receptor-ligand complexes that are internalized into clathrin-coated vesicles. Insight into what distinguishes receptors that are endocytosed from those that are not has come from studies with mutant receptors. For example, mutant forms of the LDL receptor protein are available from persons who have the inherited disorder familial hypercholesterolemia. This disease is characterized by high levels of cholesterol in the blood, and persons homozygous for the mutant alleles often die at an early age from heart attacks caused by atherosclerosis, a buildup of cholesterol deposits that ultimately block the arteries.

In some persons with this disorder, the LDL receptor is simply not produced; in others, it binds LDL poorly or not at all. In one especially instructive case, the mutant receptor binds LDL normally but the LDL-receptor complex cannot be internalized by the cell and is distributed evenly over the cell surface rather than confined to clathrin-coated pits. In individuals with this particular defect, plasma-membrane receptors for other ligands are internalized normally in clathrin-coated pits, but the mutant LDL receptor apparently cannot bind properly to coated pits. The mutant receptor has a single tyrosine-to-cysteine change in its cytosolic domain.

This and other mutant forms of the LDL receptor have been experimentally generated and expressed in fibroblasts. Analysis of these proteins has shown that a four-residue sequence in the cytosolic domain is crucial for internalization: Tyr-X-X-ø, where X can be any amino acid and ø is a bulky hydrophobic amino acid, such as Phe, Leu, or Met. As we discuss later, this sequence binds to one of the subunits (μ2) of the protein complex that links the clathrin coat to the cytosolic domain of a membrane protein in forming coated pits. Because the tyrosine and ø residues mediate this binding, a mutation in either one reduces or abolishes the ability of the receptor to be incorporated into clathrin-coated pits.

Many other plasma-membrane receptors that are internalized into clathrin-coated pits, such as the transferrin receptor, contain a similar amino acid sequence in their cytosolic domains. Mutagenesis studies on these receptors have confirmed that these four amino acids form a general recognition signal for binding to clathrin-coated pits. As further evidence for the importance of this sequence, a cell-surface protein that is not normally internalized into clathrin-coated pits can be made to internalize if these four amino acids are added to its cytosolic domain. For example, a mutant influenza HA protein, genetically engineered to contain such a four-amino-acid recognition sequence in its cytosolic domain, is internalized into clathrin-coated pits.

However, in other proteins different amino acids sequences, such as Leu-Leu, signal endocytosis (Table 17-6). Yet other membrane proteins, such as the yeast α factor receptor, uracil permease, and the human growth hormone receptor require covalent addition of ubiquitin to their cytosolic domain for endocytosis to occur. At present we do not know how endocytosis of such proteins is controlled, nor the identity of the proteins that might bind to these signals.

The Acidic pH of Late Endosomes Causes Most Receptors and Ligands 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 phospholipids each hour. Most cell-surface receptors that undergo endocytosis will repeatedly deposit their ligands within the cell and then recycle to the plasma membrane, once again to mediate the internalization of ligand molecules. For instance, the LDL receptor makes one round trip into and out of the cell every 10 – 20 minutes, for a total of several hundred trips in its 20-hour life span. In contrast, after binding its protein ligand the receptors for insulin and other growth factors generally cycle only two or three times before the complex of receptor and ligand is degraded in the lysosome — reducing the number of cell-surface receptors and thus the sensitivity of the cells to hormone signaling.

Regardless of how many times a particular receptor is recycled, internalized receptor-ligand complexes commonly follow the pathway depicted in Figure 17-46. Endocytosed cell-surface receptors dissociate from their ligands within late endosomes. These acidic spherical vesicles with tubular branching membranes are found a few micrometers from the cell surface. (Similar acidic sorting vesicles recycle M6P receptors back to the Golgi complex; see Figure 17-40.)

The original experiments that defined the late endosome sorting vesicle utilized the asialoglycoprotein receptor. This liver-specific protein mediates the binding and internalization of abnormal glycoproteins whose oligosaccharides terminate in galactose rather than the normal sialic acid, hence the name asialoglycoprotein. Electron microscopy of liver cells perfused with asialoglycoprotein reveal that between 5 and 10 minutes after internalization both the receptors and their ligands accumulate in late endosome vesicles (Figure 17-47). Ligand molecules are found in the lumen of the spherical part of these vesicles, while the tubular membrane extensions are rich in receptor and rarely contain ligand. Thus these membranes contain receptors that have dissociated from their ligands, indicating that the late endosome is the organelle in which receptors and ligands are uncoupled.

Beginning 15 minutes after internalization, ligands are transferred to lysosomes, but the intact receptors themselves usually are not found in these organelles. Instead, the receptor-rich elongated membrane vesicles that bud from the late endosomes mediate the recycling of receptors back to the cell surface (see Figure 17-46). The spherical part of the late endosome eventually buds off transport vesicles that, with their cargo of ligand, soon fuse with lysosomes. The LDL receptor, for example, is never directed to a lysosome — to be degraded by potent lysosome proteases — until it becomes damaged in some way.

The key to why receptors release their ligands in the late endosome lies in the progressively decreasing pH encountered by internalized receptor-ligand complexes as they move through clathrin-coated vesicles and various early and late endosomes. Like the very acidic lysosomes, with an internal pH of ≈4.5– 5.0, clathrin-coated vesicles and endosomes contain a V-class ATP-dependent proton pump (see Figure 15-10). These vesicles also contain a Cl⁻ channel, allowing the proton pump to generate a significant H⁺ concentration gradient, rather than the transmembrane electric potential that would form if only protons were transferred from the cytosol to the vesicle lumen. Most receptors, including the asialoglycoprotein, insulin, and LDL receptors, bind their ligands tightly at neutral pH but release their ligands if the pH is lowered to 5.0 or below. The late endosome is the first vesicle encountered by receptor-ligand complexes with a pH this low and hence is the organelle in which these and most other receptors dissociate from their tightly bound ligands.

The Endocytic Pathway Delivers Transferrin-Bound Iron to Cells

The endocytic pathway involving the transferrin receptor and its ligand differs from the LDL pathway in that the receptor-ligand complex does not dissociate in late endosomes. Nonetheless, changes in pH also mediate the sorting of receptors and ligands in the transferrin pathway, which functions to deliver iron to cells.

Transferrin, a major glycoprotein in the blood, transports iron to all tissue cells from the liver (the main site of iron storage in the body) and from the intestine (the site of iron absorption). The iron-free form, apotransferrin, binds two Fe³⁺ ions very tightly to form ferrotransferrin. All growing cells contain surface transferrin receptors that avidly bind ferrotransferrin at neutral pH, after which the receptor-bound ferrotransferrin is subjected to endocytosis. Like the components of LDL, the two bound Fe³⁺ atoms remain in the cell, but there the similarity with the fate of other endocytosed ligands, including LDL, ends: the apotransferrin part of the ligand is secreted from the cell within minutes, carried in the bloodstream to the liver or intestine, and reloaded with iron.

As depicted in Figure 17-48, the explanation for the behavior of the transferrin receptor – ligand complex lies in the unique ability of apotransferrin to remain bound to the transferrin receptor at the low pH (5.0 – 5.5) of late endosomes. At a pH of less than 6.0, the two bound Fe³⁺ atoms dissociate from ferrotransferrin and are transported from the late endosome vesicle into the cytosol (in an unknown manner). The apotransferrin formed by the dissociation of the iron atoms remains bound to the transferrin receptor and is recycled back to the surface along with the receptor. Remarkably, although apotransferrin binds tightly to its receptor at a pH of 5.0 or 6.0, it does not bind at neutral pH. Hence the bound apotransferrin dissociates from its receptor when the recycling vesicles fuse with the plasma membrane and the receptor-ligand complex encounters the neutral pH of the extracellular interstitial fluid or growth medium. The surface receptor is then free to bind another molecule of ferrotransferrin.

Some Endocytosed Proteins Remain within the Cell

In several receptor-ligand systems, found mainly in oocytes (egg cells), endocytosed material simply remains in the cells and is minimally processed. Developing insect and avian oocytes, for example, internalize yolk proteins and other proteins from the blood or surrounding cells. (Coated pits were first discovered in insect eggs, where they occupy a large portion of the plasma membrane.) A hen’s egg is a single cell containing several grams of protein, virtually all of which is imported from the bloodstream by receptormediated endocytosis. Vitellogenin, a precursor of several yolk proteins, is synthesized by the liver and secreted into the bloodstream, from which it is endocytosed into the developing egg. Yolk proteins remain in storage granules within the egg and are used after fertilization as a source of amino acids and energy by the developing embryo. Egg-white proteins (e.g., ovalbumin, lysozyme, and conalbumin) that are secreted by cells lining the hen oviduct also are endocytosed by the egg cell.

Transcytosis Moves Some Ligands across Cells

As noted previously, transcytosis is used by some cells in the apical-basolateral sorting of certain membrane proteins (see Figure 17-43). This process of transcellular transport, which combines endocytosis and exocytosis, also can be employed to import an extracellular ligand from one side of a cell, transport it across the cytoplasm, and secrete it from the plasma membrane at the opposite side. Transcytosis occurs mainly in sheets of polarized epithelial cells. An example of transcytosis is the movement of maternal immunoglobulins (antibodies) across the intestinal epithelial cells of the newborn mouse and human. The F_c receptor that mediates the transcytosis of immunoglobulins has the property of binding to its ligand at an acidic pH of 6 but not at neutral pH. Figure 17-49 shows how a difference in the pH of the extracellular media on the two sides of intestinal epithelial cells in newborn mice allows immunoglobulins to move in one direction — from the lumen to the blood. The same process also moves maternal immunoglobulins across mammalian yolk-sac cells into the fetus.

SUMMARY

• Some extracellular ligands that bind to specific cell-surface receptors are internalized, along with their receptors, in clathrin-coated vesicles. Internalized receptor-ligand complexes undergo various fates.

• Many receptors, such as the LDL receptor, release their ligand in the acidic milieu of the late endosome; the receptors are sorted into vesicles that recycle them to the plasma membrane, while the ligands are sorted into vesicles that fuse with lysosomes (see Figure 17-46). In this endocytic pathway, the released ligands are degraded by lysosomal enzymes.

• Studies with mutant LDL receptors in humans with familial hypercholesterolemia revealed a Tyr-X-X-ø signal for internalizing receptors into clathrin-coated pits. Other membrane proteins contain different endocytosis signals.

• Unlike the LDL receptor, the transferrin receptor does not release its ligand following endocytosis. Rather, the two iron atoms bound to ferrotransferrin are released in the acidic late endosome, but the resulting apotransferrin remains tightly bound to its receptor and is recycled back to the plasma membrane (see Figure 17-48). At the neutral pH on the cell surface, aprotransferrin is released from the receptor.

• In several receptor-ligand systems, found mainly in oocytes, endocytosed yolk proteins and other proteins remain in the cells and are minimally processed.

• In transcytosis, endocytosed material passes all the way through the cells and is exocytosed from the plasma membrane at the opposite side. An example is the movement of maternal immunoglobulins across mammalian yolk-sac cells into the fetus and across the intestinal epithelial cells of the newborn mouse.