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Lecture 6

Lecture 6 - Receptor-Mediated Endocytosis

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Western University
Biology 2382B
Sashko Damjanovski

LECTURE 6: RECEPTOR-MEDIATED ENDOCYTOSIS How can cells internalize the extracellular materials? • Phagocytosis o Anonselective actin-mediated process in which extensions of the plasma membrane envelop the ingested material, forming large vesicles called phagosomes o Macrophages are important for recognizing foreign pathogens, encapsulating them, breaking them apart, thus killing bacteria • Pinocytosis o Very small endocytic vesicles that are formed in which there is slight indentation in the plasma membrane that takes in some fluid outside of the cell – some substances get caught up in the process o Aform of endocytosis in which small droplets of extracellular fluid and any mater dissolved in it are nonspecifically taken up • Receptor-mediated endocytosis o Amethod of selective internalization of specific extracellular molecules (ligands)  LDL(low-density lipoprotein)  Transferrin  Hormones (e.g. insulin), certain glycoproteins, etc. o Aspecific 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. Low-density lipoprotein (LDL) • Lipids are transported in large well-defined water-soluble complexes/particles called lipoproteins • LDLcontain approx. 88% cholesteryl esters and mediate cholesterol transport • LDLreceptors on plasma membrane (localized in clathrin-coated pits) • Amphipathic shell, composed of a phospholipid monolayer, and apolipoprotein • Apolar core – hydrophobic, mostly cholesteryl esters • To facilitate the mass transfer of lipids between cells, animals have evolved an efficient way to package from hundreds to thousands of lipid molecules into water-soluble, macromolecular carriers, called lipoproteins, that cells can take up from the circulation as an ensemble • Alipoprotein particle has a shell composed of proteins (apolipoproteins) and a cholesterol-containing phospholipid monolayer • The shell is amphipathic because its outer surface is hydrophilic, making these particles water soluble, and its inner surface is hydrophobic • Adjacent to the hydrophobic inner surface of the shell is a core of neutral lipids containing mostly cholesteryl esters, triglycerides or both • LDLis important for moving lipids around • Lipids are hydrophobic so they won’t transport well unless incorporated in something that facilitates transferring in an aqueous environment • LDLparticles are lipid cores containing mainly cholesteryl esters • Hydrocarbon chains face the inside; outside is hydrophilic – stable in aqueous environment and inside is hydrophobic and can bind to cholesterol • Apolipoprotein B – basically a protein that facilitates interaction between LDLparticles • LDLparticles are transported in blood • ApoB binds to receptors – this process helps promote an endocytic event associated with Clathrin Electron Microscopy Data • Receptors in plasma membrane recognizes a unique ligand (LDL) • Clathrin helps internalize ligand • Researchers took LDLand linked it to ferritin (an iron-binding protein) – an artificially created situation to understand how LDL enters cells • LDL-ferritin conjugates were added to cell culture (human fibroblasts) • Processed for transmission EM, start to see little specks of LDL-ferritin complexes • Stain allows to visualize Clathrin-coat protein pits • Association of LDL particles at plasma membrane, accumulation of Clathrin molecules • Lead to invagination – called this Clathrin-coated pits • Particles later get incorporated into endocytic vesicles, Clathrin is still in association • Endocytic vesicles have Clathrin on the outside, LDL-ferritin labeled particles on the inside (early endosome) • Vesicle becomes fully detached from membrane Clathrin/AP-Coated Vesicles • Two layer coat: clathrin and adapter protein (AP) complexes: o AP1 – TGN o AP2 - PM • AP complexes recognize sorting signals of cargo proteins or receptors. • Clathrin-coated vesicles pinch off using dynamin and GTP hydrolysis (dynamin forms rings around the vesicle neck, polymerizes) • LDLparticle binds to LDLreceptor; when bound, a conformational change is triggered and results in sorting signals being exposed at the cytoplasmic domain • Binding of LDLto LDLreceptor causes sorting signals to recruit coat proteins, which will associate locally at the plasma membrane, and promote association of Clathrin • Will associateAP2 and Clathrin accumulation • Promote invagination of plasma membrane, but needs help pinching off • Dynamin, with the help of GTP that will be hydrolyzed to GDP, can make pinching off occur and the endocytic vesicle that is Clathrin-coated can be formed • AP2 is involved, need Clathrin for this process – need to pinch off vesicle from membrane and need dynamin for this, which can hydrolyze GDP pH-dependent binding of LDLparticles to the LDLreceptor • LDLR has three domains: o Short C-terminal cytosolic segment with sorting signal o Long N-terminal exoplasmic segment with a ligand binding domain and b-propeller domain • At normal pH the ligand-binding arm binds tightly toApoβ. • At acidic pH 5.0-5.5, histidine residues in β-propeller domain become protonated and bind with high affinity to the negatively charged residues in the ligand-binding arm • β-propeller domain has histidine residues o At an acidic pH, they become protonated (positively charged) o Ligand-binding arm will be positive and tucks in, releasing the LDL particle (this takes place in the endosome) Targeting LDLand LDLreceptors to clathrin/AP2-coated pits • Specific sorting signals in the cytoplasmic domain (a four-residue motif) of receptors binds the AP2
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