Clathrin-coated vesicle receptor endocytosis

Most proteins that are synthesized on membrane-bound polyribosomes and are destined for the Golgi apparatus or plasma membrane reach these sites inside transport vesicles. The precise mechanisms by which proteins synthesized in the rough ER are inserted into these vesicles are not known. Those involved in transport from the ER to the Golgi apparatus and vice versa—and from the Golgi to the plasma membrane— are mainly clathrin-free, unlike the coated vesicles involved in endocytosis (see discussions of the LDL receptor in Chapters 25 and 26). For the sake of clarity, the non-clathrin-coated vesicles will be referred to in... 

Extracellular ligands (hormones, neurotrophins, carrier protein, adhesion molecules, small molecules, etc.) will bind to specific transmembrane receptors. This binding of specific ligand induces the concentration of the receptor in coated pits and internalization via clathrin-coated vesicles. One of the best studied and characterized examples of RME is the internalization of cholesterol by mammalian cells [69]. In the nervous system, there are a plethora of different membrane receptors that bind extracellular molecules, including neurotrophins, hormones and other cell modulators, being the best studied examples. This type of clathrin-mediated endocytosis is an amazingly efficient process, capable of concentrating... 

Receptor-mediated endocytosis is the selective uptake of extracellular macromolecules (such as cholesterol) through their binding to specific cell-surface receptors. The receptor-macromolecule complex then accumulates in clathrin-coated pits and is endocytosed via a clathrin-coated vesicle. 

In contrast, receptor-mediated endocytosis is very specific. Embedded in the membrane are proteins with specific receptor sites exposed to the extracellular fluid. The receptor proteins are usually clustered in regions of the membrane called coated pits, which are lined on their cytoplasmic side by a fuzzy layer consisting of a protein called clathrin. When ligands bind to the receptor sites, they are carried into the cell by the inward budding of a coated pit to form a coated vesicle. Clathrin-coated vesicles become uncoated and fuse to form an endosome. Ligand and receptor dissociate within the endosome, and the receptor shuttles back to the cell surface. The endosome fuses with the lysosome on which ligand degradation occurs. 

The work reviewed here has begun to provide a detailed picture of the biosynthesis and cellular processing of PrP and PrP. PrP is synthesized and matures along the secretory pathway much like other membrane glycoproteins. One important feature is the addition of a GPI anchor that serves to attach the polypeptide chain to the lipid bilayer without the necessity for a transmembrane domain. Once it reaches the plasma membrane, PrP is constitutively endocytosed via clathrin-coated vesicles, and a portion of the molecules is proteolyti-cally cleaved in a highly conserved domain before recycling to the cell surface. Efficient endocytosis depends on structural features in the N-terminal half of the PrP molecule and may be mediated by a transmembrane PrP receptor . 

Fig. 3. Model of the life cycle of prions. PrP is synthesized in the rough endoplas-matic reticulum (ER), and after passing through the secretory pathway including the Golgi and secretory vesicles, reaches the surface of a PrP infectable cell where it is anchored via a glycosylphosphatidyl inositol (GPI) moiety. Endocytosis of PrP and possibly PrP via clathrin coated vesicles could be mediated by the 37 kDa laminin receptor precursor (LRP). The uptake of the infectious agent could also be LRP independent. The conversion of the internalized PrP to PrP is thought to take place in the endo-somes, lysosomes, or endolysosomes. Molecular chaperones could be involved in this conversion process. PrP replication and aggregation can occur in neuronal cells of the brain but also in the cells constituting the lymphoreticular system. Alternatively, endocytosis and conversion of PrP into PrP could happen in caveolae-like domains (CLDs).

Vesicular transport occurs when a membrane completely surrounds a compound, particle, or cell and encloses it into a vesicle. When the vesicle fuses with another membrane system, the entrapped compounds are released. Endocytosis refers to vesicular transport into the cell, and exocytosis to transport out of the cell. Endocytosis is further classified as phagocytosis if the vesicle forms around particulate matter (such as whole bacterial cells or metals and dyes from a tattoo), and pinocy-tosis if the vesicle forms around fluid containing dispersed molecules. Receptor-mediated endocytosis is the name given to the formation of clathrin-coated vesicles that mediate the internalization of membrane-bound receptors in vesicles coated on the intracellular side with subunits of the protein clathrin (Eig. 10.14). Potocytosis is the name given to endocytosis that occurs via caveolae (small invaginations or caves ), which are regions of the cell membrane with a unique lipid and protein composition (including the protein caveolin-1). 

F. 10.14. Formation of a clathrin-coated vesicle. Ligands entering the cell through receptor-mediated endocytosis bind to receptors that cluster in an area of the membrane. Adaptor proteins bind to the receptor tails and to the clathrin molecules to enclose the budding membrane in a cage-like clathrin coat. 

The best-documented way of endocytosis is receptor-mediated uptake of ligands via clathrin-coated vesicles (reviewed in Schmid 1997). Receptors are recruited and concentrated into clathrin-coated pits at the plasma membrane. After coated vesicle formation, the clathrin coat is removed by the concerted action of auxihn and heat shock protein 70 (Ungewickell et al. 1995). The uncoated vesicles fuse with early endosomes in a rab5-regulated manner (Rubino et al. 2000). 

Figure 41 -15. Two types of endocytosis. An endocytotic vesicle (V) forms as a result of invagination of a portion of the plasma membrane. Fluid-phase endocytosis (A) is random and nondirected. Receptor-mediated endocytosis (B) is selective and occurs in coated pits (CP) lined with the protein clathrin (the fuzzy material). Targeting is provided by receptors (black symbols) specific for a variety of molecules. This results in the formation of a coated vesicle (CV).

Retrieval of membrane components in the secretory pathway through receptor-mediated endocytosis (RME) is a clathrin-coat-dependent process [5]. The clathrin coat provides stability to the vesicle core and allows uptake of specific membrane proteins for reuse or degradation. RME shows a remarkable degree of specificity, allowing cells to internalize with astonishing efficiency only those selected molecules independent of their extracellular concentration. 

Clathrin-mediated endocytosis involves the internalization of transmembrane receptor-ligand complexes stimulating the formation of a coated pit that eventually buds off the membrane to form an intracellular endocy-totic vesicle. This process is dependent on the protein clathrin that is recruited to the membrane and forms a cage-like structure around the forming pit. Internalization via clathrin-dependent pathway allows the uptake of particles approximately 120nm in size (63-65). Once internalized, the clathrin coating disassociates from the endosome to be recycled and to allow the endosome to fuse with an intracellular compartment, usually a... 

VLDLs, IDLs, and LDLs are closely related to one another. VLDLs formed in the liver (see p. 312) transport triacylglycerols, cholesterol, and phospholipids to other tissues. Like chylomicrons, they are gradually converted into IDL and LDL under the influence of lipoprotein lipase [1]. This process is also stimulated by HDL. Cells that have a demand for cholesterol bind LDL through an interaction between their LDL receptor and ApoB-100, and then take up the complete particle through receptor-mediated endocytosis. This type of transport is mediated by depressions in the membrane ( coated pits"), the interior of which is lined with the protein clathrin. After LDL binding, clathrin promotes invagination of the pits and pinching off of vesicles ( coated vesicles"). The clathrin then dissociates off and is reused. After fusion of the vesicle with ly-sosomes, the LDL particles are broken down (see p. 234), and cholesterol and other lipids are used by the cells. 

Receptor clustering also accounts for receptor internalization. The basis of this phenomenon is endocytosis via coated pits. These pits are apparent in electron micrographs as membrane invaginations coated on the inner (cytoplasmic) side with a web of the protein clathrine. It has been suggested that certain receptor proteins have structural domains that allow them to react with coated pits. Receptor clusters in coated pits are rapidly endocytosed, resulting in the formation of vesicles (endosomes) that are then... 

Fig. 3. Receptor-mediated endocytosis involves clathrin-coated pits and vesicles.

Gene delivery systems can distribute plasmids to the desired target cells, after which the plasmid is internalized into the cell by a number of mechanisms, such as adsorptive endocytosis, receptor-mediated endocytosis, micropinocytosis, caveolae-mediated endocytosis and phagocytosis (see Section 1.3.3.2). The intracellular fate of plasmids depends on the means by which they are internalized and translocated to the cytoplasms and then to the nucleus. In coated-pit endocytosis, DNA complexes first bind to the cell surface, then migrate to clathrin-coated pits about 150 ran in diameter and are internalized from the plasma membrane to form coated vesicles. 

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