9 Receptor-Mediated Endocytosis|Advanced Cell Biology|Tulane

Receptor-Mediated Endocytosis

Influenza Virus Infects by Viral Hemagglutinin Interaction with Host Cell Sialic Acid

© Creative Biolabs

Host Antigen: Sialic acid; Viral Antigens: Hemagglutinin (H), Neuraminidase (N)
Neuraminidase cleaves host Sialic acid, enhances release of new viral particles.
Antiviral Neuraminidase inhibitors: Tamiflu (Oseltamivir) and Relenza (Zanamivir). Viral subtypes derive from 18 H and 11 N possible antigens. Vaccine against common H and N antigens. Changes yearly due to antigenic drift.

Endocytosis activate: [Hemagglutinin]-[Sialic]
Lysosome Fuse: H⁺ release to change the pH → Hemagglutinin conformation changed
Neuraminidase： Cleave Sialic → Enhance releases
Antiviral Neuraminidase inhibitors: Tamiflu (Oseltamivir) and Relenza (Zanamivir).

© Dr.G Bhanu Prakash Animated Medical Videos; 2019 youtube

14.5 Receptor-Mediated Endocytosis

• Clathrin-coated vesicles: [Ligands]::[AP2-targeting sequences]-[Receptors]
• Ligands delivers: Exp: LDL
• Dissociation: Receptor-ligand complexes dissociated in acid environment:
  • receptor → cytoplasm → recycling
  • ligand → lysosome → degradation
• Ttransferrin carrier: Iron endocytosis Fe³⁺ → endosome and recycle transferrin

• Extracellular ligands bound to specific cell-surface receptors with cytoplasmic domain AP2-targeting sequences are internalized by clathrin-coated vesicles.
• The endocytic pathway delivers some ligands (e.g., LDL particles) to lysosomes, where they are degraded.
• The late endosome acidic environment dissociates most receptor-ligand complexes for receptor recycling to the plasma membrane and ligand degradation in lysosomes.
• The iron endocytosis pathway releases Fe3+ in the late endosome but recycles the transferrin carrier proteins with the receptor to the plasma membrane.

Initial Stages of RME of Low-Density Lipoprotein (LDL) Particles

© Lodish, Molecular Cell Biology, Eight Edition, p661](https://www.amazon.com/Molecular-Cell-Biology-Harvey-Lodish/dp/1464183392)	(a) LDL-LDL receptor complexes invaginated in AP2-clathrin-coated pit. (b) Pit closes. Dynamin associates with neck region. © AP2-Clathrin-coated vesicle containing free in cytoplasm (d) LDL-LDL receptor complexes in decoated endosome.

© iotech Review; 2014 youtube

RME: Receptor-mediated endocytosis

LDL: low-density lipoprotein

• RME is used by cells to import specific macromolecules or complexes too large to be imported by membrane transporters
• Uptake specificity is receptor-dependent, in clathrin/AP2-coated vesicles.

Experiment:

1. LDL particles (contain cholesterol): labeled with electron-dense, iron-containing ferritin protein (visible with EM)
2. Cultured fibroblasts incubated with LDL-ferretin at 4°: LDL binds to LDL-receptors; endocytosis process is inhibited
3. Cells warmed to initiate endocytosis.

RME receptors:

• Some types cluster in clathrin-coated pits by cytoplasmic domain association with AP2 even in absence of ligand.
• Others types diffuse freely in the plasma membrane until a ligand-induced conformational change associates them with AP2.
• Two or more types of receptor-bound ligands, such as LDL and transferrin, can be present in the same coated pit/vesicle.

Cellular Uptake / Degradation of LDL by RME

© Lodish, Molecular Cell Biology, Eight Edition, p661

• LDL-LDL receptor complexes completely bound at membrane surface in AP2-clathrincoated pit.
• Internalization of LDL-LDL receptor receptor increases as binding at cell surface decreases.
• Degradation begins within 10-15 minutes, as internalization reached its maximum.

Pulse-chase experiment demonstrates precursor-product relations in cellular uptake of LDL.
Experiment:

1. LDL labeled with ¹²⁵I
2. Cultured normal human skin fibroblasts – incubated with ¹²⁵I-LDL for 2 hours at 4°C (pulse) – LDL binds to surface LDL receptors; not endocytosed
3. Unbound LDL – washed away
4. Shift to 37°C – activates RME (chase)
5. Results:
   • ¹²⁵I-LDL rapidly disappears from surface (binding) by RME.
   • ¹²⁵I-LDL in internal vesicles rises coincidently.
   • After a 15 min lag – ¹²⁵I-LDL degradation in lysosomes increases.

Model of LDL Particle

© Lodish, Molecular Cell Biology, Eight Edition, p661

• Cells take up lipids from the blood in the form of large, welldefined lipoprotein complexes (LDL, VLDL, HDL, Chylomicon).
• All classes of lipoproteins have the same general structure. Model of LDL Receptor and LDL Binding

• Shell composed of a phospholipid monolayer (not bilayer) containing cholesterol. Interacts with aqueous environment for transport in blood.
• Apolipoprotein B (ApoB) is ligand for LDL receptor.
• Hydrophobic core is mostly cholesteryl esters/triglycerides (minor neutral lipids [vitamins]).

Model of LDL Receptor and LDL Binding

© Lodish, Molecular Cell Biology, Eight Edition, p663

(a) LDL receptor at neutral pH (binding LDL at cell surface):
- Ligand binding arm seven cysteine-rich repeats (R1–R7) – tightly bind LDL apoB-100 (R4 and R5 – most critical for LDL binding)
- Note receptor NPXY AP2- targeting cytosolic domain.
(b) LDL receptor at acidic pH (releasing LDL in endosome):
- β-propeller domain histidine residues become protonated.
- Positively charged propeller domain binds negatively charged ligand-binding domain → release of LDL particle.
PS: (b, bottom) LDL receptor at pH 5.3 structure – extensive hydrophobic and ionic interactions between the β propeller and R4 and R5 repeats
- Human disease – Familial Hypercholesterolemia
- Excessive circulating LDL causes cardiovascular disease.
- LDL receptor mutations cause too little LDL uptake/clearance into cells (liver cells).
- Several LDL receptor mutations including a single amino acid mutation in NPXY targeting sequence causes inefficient LDL receptor RME – FH disease.

LDL-LDL Receptor Mediated Endocytosis

© Lodish, Molecular Cell Biology, Eight Edition, p662

Physiologic pH: LDL receptor has high affinity for LDL.
Lysosomal pH: LDL receptor has low affinity for LDL.
Receptor recycled

Clinical Implications: Familial Hypercholesterolemia

1. Excessive circulating LDL causes cardiovascular disease.
   a. Heterozygous – LDL ~2X elevated in blood
   b. Homozygous – LDL 4X-6X elevated
   c. Atherosclerotic plaques
   b. Premature heart attacks (as early as 20s)
2. LDL receptor mutations cause too little LDL uptake or clearance into cells (liver cells).
   a. LDL receptors are absent
   b. Defective LDL binding sites
   c. Improper folding → premature receptor degradation
   d. NPXY mutation affects sorting to AP2 vesicles
   e. Mutation in ApoB ligand, ↓ affinity for LDL receptor
3. Cells regulate their cholesterol by
   a. Inhibition of cholesterol synthesis
   b. Down-regulation of LDL receptor

Transferrin Cycle

© Lodish, Molecular Cell Biology, Eight Edition, p664

• Transferrin Cycle:

  • Ferrotransferrin binds transferrin receptor at cell surface pH 7.0 (1).
  • Transferrin receptor forms AP2 clathrin-coated vesicle → RME to late endosome (2-4).
  • Acidic pH dissociates Fe3+ → Apotransferrin recycled to cell surface.
  • Extracellular neutral pH, receptor releases apotransferrin.
  • Fe3+ reduced to Fe2+ for use.

• Transferrin protein comes in two forms, apotransferrin (not bound to Fe3+) and Ferrotransferrin (carries Fe3+ in blood).

• Transferrin binding to transferrin receptor is pH-dependent:

  • Receptor binds Ferrotransferrin at physiologic pH (pH ~7.0).
  • Receptor binds Apotransferrin at lysosomal pH (pH ~5.0).

RME endocytic pathway delivers iron to cells without dissociation of the transferrin–transferrin receptor complex in endosomes.
Transferrin protein:

• Apotransferrin – no bound Fe3+
• Ferrotransferrin – carries Fe3+ in blood
• Binds to transferrin receptor

Uptake mechanism:

1. Ferrotransferrin dimer with two Fe3+ binds tightly to transferrin receptor at the cell surface – ~pH 7.0.
2. Transferrin receptor cytoplasmic tail interaction with an AP2 adapter complex – incorporates receptor-ligand complex into endocytic clathrin-coated vesicles
3. Clathrin disassembly uncoats vesicle.
4. Vesicle fuses with late endosome – acidic pH – Dissociates Fe3+ from ferrotransferrin – Fe3+ reduced to Fe2+ by endosome reductase Transported from endosome to cytoplasm Maintains apotransferrin-receptor complex
5. Recycling of receptor-apotransferrin complex to the plasma membrane
6. Extracellular neutral pH destabilizes complex – releases apotransferrin from receptor

14.6 Directing Membrane Proteins and Cytosolic Materials to the Lysosome

ESCRT (endosomal sorting complex required for transport)

• Endocytosed membrane proteins targeted for degradation in the lysosome are incorporated into vesicles that bud into the interior of the endosome.
• Cellular components (e.g., ESCRT) that mediate endosome membrane budding are used to pinch off enveloped viruses such as HIV from the plasma membrane of virus-infected cells.
• Autophagy envelopes a region of cytoplasm or an organelle into a double-membrane autophagosome for delivery to a lysosome.

Delivery of Plasma-Membrane Proteins to Lysosome Interior for Degradation

Membrane proteins for degradation → delivered to lysosome lumen

© Lodish, Molecular Cell Biology, Eight Edition, p666

Mechanism:

1. Vesicles carrying newly synthesized lysosomal membrane proteins (green) from the trans-Golgi network – fuse with the late endosome
2. Endosomes carrying endocytosed plasma-membrane proteins (blue) targeted for degradation – fuse with the late endosome
3. Late endosome:
   • Plasma membrane proteins targeted for degradation – incorporated into vesicles that bud into the interior of the late endosome
   • Forms a multivesicular endosome
4. Multivesicular endosome fuses with a lysosome:
   • Internal vesicles (containing targeted membrane proteins)– degraded
   • Lysosomal membrane proteins – not degraded

Formation of Multivesicular Endosomes

© Lodish, Molecular Cell Biology, Eight Edition, p667

• Proteins for multivesicular endosome degradation are tagged with ubiquitin at the plasma membrane, in the trans-Golgi network, or in the endosomal membrane (cargo proteins).
• Ubiquitinated Hrs protein on endosomal membrane direct loading of ubiquitinated membrane cargo proteins (blue) into multivesicular endosomes.
• Cytosolic ESCRT protein complexes and Hrs mediate pinching off of inwardly budding vesicles.
• Vps4 ATP hydrolysis drives disassembly and recycling of ESCRT complex proteins.

HIV Budding from Plasma Membrane

© Lodish, Molecular Cell Biology, Eight Edition, p668

• Retrovirus (HIV) budding from plasma membrane exploits ESCRT/Vps4 machinery for multivesicular endosomes.
• Ubiquitinated viral Gag proteins function like Hrs protein → recruit ESCRT/Vps4 complexes to pinch off viral particle. The Autophagic Pathway
• Autophagic pathway delivers cytosolic proteins and organelles to lysosomes for degradation.
• ATG proteins induce formation of cup-shaped membrane structure around a portion of the cytosol (right) or an organelle (left) to create am autophagosome.
• Autophagosome envelop contents in two complete membranes.
• Fusion of autophagosome outer membrane with the lysosome membrane releases a singlemembrane vesicle and its contents into the lysosome interior for degradation.

Mechanism:
ATG proteins induce formation of cup-shaped membrane structure around

• (right) a portion of the cytosol
• (left) an organelle such as a mitochondrion
• Proteins involved include Atg 5, 8, 12, 16 – Atg 8 forms coat around autophagosome

Step 1: Continued membrane addition and fusion forms autophagosome – envelops contents in two complete membranes
Step 2: Fusion of the autophagosome outer membrane with the lysosome membrane –

• Releases a single-membrane vesicle and its contents into the lysosome interior
• Vesicle protein and lipid components – degraded by lysosomal hydrolases
• Amino acids – permease transport across the lysosomal membrane into the cytosol for use in protein synthesis