Transport mechanisms receptor-mediated endocytosis

An important first step in the hepatic metabolism of proteins and peptides is the uptake into hepatocytes. Small peptides may cross the hepatocyte membrane via passive diffusion if they have sufficient hydrophobicity. Uptake of larger peptides and proteins is facilitated via various carrier-mediated, energy-dependent transport processes. Receptor-mediated endocytosis is an additional mechanism for uptake into hepatocytes (see Sect. 8.3.4.5) [28]. In addition, peptides such as metkephamid can already be metabolized on the surface of hepatocytes or endothelial cells [41]. 

Huang and Swaan (2000) revealed that the receptor-mediated mechanism of riboflavin takes place in the nanomolar range of riboflavin concentrations, whereas carrier-mediated process dominates in the micromolar range. They observed the inhibition of basolateral-apical transport of riboflavin by the same substances as transferrin, which is the typical protein transported through receptor-mediated endocytosis, i.e. brefeldin A and nocodazole. Brefeldin A strongly increased apical to basolateral riboflavin transport, while nocodazole increased apical to basolateral and inhibited basolateral to apical flux of riboflavin. 

Figure 11.1 Schematic representation of iron uptake mechanisms, (a) The transferrin-mediated pathway in animals involves receptor-mediated endocytosis of diferric transferrin (Tf), release of iron at the lower pH of the endocytic vesicle and recycling of apoTf. (b) The mechanism in H. influenzae involves extraction of iron from Tf at outer membrane receptors and transport to the inner membrane permease system by a periplasmic ferric binding protein (Fbp). From Baker, 1997. Reproduced by permission of Nature Publishing Group.

Lipids are transported between membranes. As indicated above, lipids are often biosynthesized in one intracellular membrane and must be transported to other intracellular compartments for membrane biogenesis. Because lipids are insoluble in water, special mechanisms must exist for the inter- and intracellular transport of membrane lipids. Vesicular trafficking, cytoplasmic transfer-exchange proteins and direct transfer across membrane contacts can transport lipids from one membrane to another. The best understood of such mechanisms is vesicular transport, wherein the lipid molecules are sorted into membrane vesicles that bud out from the donor membrane and travel to and then fuse with the recipient membrane. The well characterized transport of plasma cholesterol into cells via receptor-mediated endocytosis is a useful model of this type of lipid transport. [9, 20]. A brain specific transporter for cholesterol has been identified (see Chapter 5). It is believed that transport of cholesterol from the endoplasmic reticulum to other membranes and of glycolipids from the Golgi bodies to the plasma membrane is mediated by similar mechanisms. The transport of phosphoglycerides is less clearly understood. Recent evidence suggests that net phospholipid movement between subcellular membranes may occur via specialized zones of apposition, as characterized for transfer of PtdSer between mitochondria and the endoplasmic reticulum [21]. 

However, the receptor-mediated endocytosis of iron-transferrin studies [63] does not explain the initial uptake of iron from nutrients in the intestinal (villus) cells, since apotransferrin is generally not available in the lumen, except in a limited amount from biliary excretion. Work on other iron transport mechanisms has mainly been reported in the last five years. 

Possible explanations for a blood flow-limited uptake in kidney include the existence of specific uptake mechanisms, such as receptor-mediated endocytosis and carrier-mediated transport. Since the former mechanism is initiated by binding of the ligand to the cell-surface receptor, the specific binding of alkylglycoside compounds to isolated tubular plasma membranes was examined [23,24]. 

HDL may be taken up in the liver by receptor-mediated endocytosis, but at least some of the cholesterol in HDL is delivered to other tissues by a novel mechanism. HDL can bind to plasma membrane receptor proteins called SR-BI in hepatic and steroidogenic tissues such as the adrenal gland. These receptors mediate not endocytosis but a partial and selective transfer of cholesterol and other lipids in HDL into the cell. Depleted HDL then dissociates to recirculate in the bloodstream and extract more lipids from chylomicron and VLDL remnants. Depleted HDL can also pick up cholesterol stored in extrahepatic tissues and carry it to the liver, in reverse cholesterol transport pathways (Fig. 21-40). In one reverse transport path, interaction of nascent HDL with SR-BI receptors in cholesterol-rich cells triggers passive movement of cholesterol from the cell surface into HDL, which then carries it back to the liver. In a second pathway, apoA-I in depleted HDL in-... 

Weissleder et al. [84] first showed that the human transferrin receptor (hTfR) can be used to internalize MRI contrast agents. The hTfR regulates cellular uptake of iron from transferrin, a plasmatic iron transport protein [85], via a receptor mediated endocytosis mechanism. Thus, MION particles (dextran coated iron oxide) were oxidized with sodium periodate. Holotransferrin was added and the resulting Schiff base adduct was reduced with sodium cyanoborohydride to give transferrin labeled MIONs, Tf-MION (Scheme 3). 

In all three cases the contents of the intracellular vesicles will finally be released into the cytoplasm, or transported to the cell membrane to be released into the ex-travascular space. Receptor-mediated endocytosis is the predominant mechanism of cellular uptake for mAbs. As mentioned previously (see Sections 3.9.1 and 3.9.3), Fc-Rn is present in a large variety of cells and is very often involved in this process. In addition, the antigen-antibody complex via Fab can also undergo re-ceptor -mediated endocytosis. The impact of this internalization process on the pharmacokinetics of mAbs will be discussed later. 

Be familiar with the composition and structure of biologic membranes. Be able to place the various phospholipids in the membrane bilayer. Know the function and position of membrane proteins and their possible movements. Know how membrane fluidity is controlled. Know the nature of various mechanisms to transport substances across membranes, receptor-mediated endocytosis, active and facilitated transport, ionophores, and the various types of channels. Be able to solve simple mathematical problems by creating solute gradients across membranes. Know the names of substances that inhibit the various modes of transport across membranes. 

Three processes are involved in transcellular transport across the intestinal epithelial cells simple passive trans-port, passive diffusion together with an efflux pump, and active transport and endocytosis. Simple passive transport is the diffusion of molecules across the membrane by thermodynamic driving forces and does not require direct expenditure of metabolic energy. In contrast, active transport is the movement of molecules across the mem-brane resulting directly from the expenditure of metabolic energy and transport against a concentration gradient. Endocytosis processes include three mechanisms fluid-phase endocytosis (pinocytosis), receptor-mediated endocytosis, and transcytosis (Fig. 6). Endocytosis processes are covered in detail in section Absorption of Polypeptides and Proteins, later. 

Fig-1 The final NM-induced toxic effect observed in vitro is the result of multiple processes (1) interaction with proteins (formation of the protein corona, activation/inactivation of enzymes) (2) dissolution and release of toxic ions (3) production of ROS at the NMs surface (4) aggregation/agglomeration (5) diffusion and sedimentation that influence NM transport to the cell layer and the final effective concentration (6) interaction with the cell membrane and membrane receptors (activation/inhibition) (7) cell uptake (including receptor-mediated endocytosis and other uptake mechanisms) (8) interaction with intracellular enzymes (activation/inhibition) (9) production of intracellular ROS (10) activation of transcription factors and (11) binding to nucleic acids and genotoxicity, among others. Processes (1)—(5) are closely interconnected. The resulting effect observed is the result of the composite rate of all these different reactions... 

Figure 8,1, Routes and mechanisms of solute transport across epithelial membranes. In general, routes 2-5 are transcellular pathways (i.e., compounds move through the cells), whereas route 1 is considered a paracellular pathway (i.e., a compound moves between the cells). (l)Tight junctional pathway (2) drug efflux pathway (e.g., P-glycoprotein mediated) (3) passive diffiision (4)receptor-mediated endocytosis and/or transc3dosis pathways (5) carrier-mediated route. Note that receptor and carrier proteins in epithelial cells are expressed on both the apical and basolateral surfaces.

Figure 18-16 depicts a model for the selective uptake of cholesteryl esters by a cell-surface receptor called SR-BI (scavenger receptor, class B, type I). SR-BI binds HDL, LDL, and VLDL and can mediate selective uptake from all of these lipoproteins. The detailed mechanism of selective llpid uptake has not yet been elucidated, but It may entail hemifuslon of the outer phospholipid monolayer of the lipoprotein and the exoplasmic leaflet of the plasma membrane. The cholesteryl esters Initially enter the hydrophobic center of the plasma membrane, are subsequently transferred across the Inner leaflet, and are eventually hydrolyzed by cytosolic, not lysosomal, cholesteryl esterases. The llpid-depleted particles remaining after llpid transfer dissociate from SR-BI and return to the circulation they can then extract more phospholipid and cholesterol from other cells by means of the ABCAl protein or other cell-surface transport proteins (see Figure 18-13c). Eventually, small llpid-depleted HDL particles circulating In the bloodstream are filtered out by the kidney and bind to a different receptor on renal epithelial cells. After these particles have been Internalized by receptor-mediated endocytosis, they are degraded by lysosomes.

Lipoproteins are soluble complexes of proteins (apolipoproteins) and lipids that transport lipids in the circulation of all vertebrates and even insects. Lipoproteins are synthesized in the liver and the intestines, arise from metabolic changes of precursor lipoproteins, or are assembled at the cell membranes from cellular lipids and exogenous lipoproteins or apolipoproteins. In the circulation, lipoproteins are highly dynamic. They undergo enzymatic reactions of their lipid components, facilitated and spontaneous lipid transfers, transfers of soluble apolipoproteins, and conformational changes of the apolipoproteins in response to the compositional changes. Finally, lipoproteins are taken up and catabolized in the liver, kidney, and peripheral tissues via receptor-mediated endocytosis and other mechanisms. This chapter deals with the composition and structure of human lipoproteins. 

The mechanism of receptor-mediated endocytosis is based on interactions between a transported substance and a special protein (receptor) bound to a cell membrane. Molecules are directly recognized by the receptor substance, or may be at first attached to a special protein, which in turn forms a complex with the receptor. The complex is locked in coated pits or vesicles, and next transported within the cytosol. The vesicles are uncoated by an ATP-dependent enzyme the complex from their core is located inside endosomes and, after its dissociation from receptors, it is further conveyed to lysosomes. 

An additional mechanism for transport of metal complexes is by endo-cytosis/exocytosis (for review see Ballatori 1991). Fluid-phase, adsorptive, and receptor-mediated endocytosis make a major contribution to the transport of metals that are bound to high molecular weight ligands, and in particular to ligands such as ferritin, transferrin, and other proteins that are selectively cleared by receptor-mediated endocytosis. Because these proteins also have some affinity for toxic metals, they may play an important role in their transport across cell membranes (Ballatori 1991). The mechanism by which metallothionein and its associated metals are removed from the circulation is not known, but the kidney appears to be the principal site of removal (Tanaka et al. 1975). When rats are injected intravenously with ° Cd-labeled metallothionein, the radioactivity is rapidly and nearly completely accumulated in the kidney (Tanaka et al. 1975). 

The sequestration of cadmium by MT is a double-edged sword, i.e., although Cd-MT is relatively inert when stored as an intracellular complex, it becomes a potent nephrotoxicant after reaching the systemic circulation (Cherian et al. 1976 Squibb et al. 1984). Human cadmium nephrotoxicity may be related to Cd-MT exposure, because this may be a major form of cadmium in diet (Maitani et al. 1984). Cadmium salts absorbed from the GI tract or lungs are initially transported to liver, where synthesis of MT is induced. Continual exposure to cadmium results in liver injury with leakage of Cd-MT into the systemic circulation (Dudley et al. 1985). The complex is transported to kidney, filtered, and reabsorbed by the proximal tubule, possibly via a mechanism involving receptor mediated endocytosis (Foulkes... 

The transferrin receptor was one of the first to be exploited for receptor-mediated gene delivery. All actively metabolising cells require iron that is internalised by the cell as a transferrin-iron complex by means of receptor mediated endocytosis. To exploit this ubiquitous and efficient transport mechanism for introducing DNA into cells, conjugates of chicken or human transferrin with polycations (polylysine or protamine) were synthesised and used to form complexes with plasmid DNA. The number of transferrin molecules attached to each polylysine molecule varies according to the molecular weight of the polymer, but is generally around 1 transferrin molecule for every 50 lysine residues. ... 

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