Receptor-Mediated Endocytosis experimental pathway （ II ）

1. Receptor-Mediated Endocytosis experimental pathway（II） Wu Chenzi

2. Experiments about … • The process of receptor binding and internalization（内化作用） • The LDL receptor • Internalization signals

3. The process ofreceptor binding and internalization Technique — electron microscope To visualize the process of receptor binding and internalization, MichaelBrown and JosephGoldstein (University of Texas Medical School in Dallas) teamed up with Richard Anderson, who had been studying cellular structure with the electron microscope.

4. Experimental pathway • The group incubated fibroblasts（成纤维细胞） from normal and FH subjects with LDL that had been covalently linked（共价结合） to the iron-containing protein ferritin（铁蛋白）. • Because of the iron atoms, ferritin molecules are able to scatter a beam of electrons and thus can be visualized in electron microscope. • A temperature of4℃ Ligands（配体） can bind to the cell surface but cannot be internalized（内化）, LDL-ferritin particles were seen to be bound to the cell surface.

5. Found that … 1、The LDL particles were not randomly scattered over the cell surface but were localized to some segmentsof plasma membrane—— coated pits. 2、Cells from patients with FH were found to have a similar number of coated pits on their surface, butno LDL-ferritin was bound to these mutant cells.

6. The results supported the proposal thatthe mutant FH allele encoded receptor that was unable to bind LDL. Subsequent electron microscopic studies on the internalization of LDL-ferritin revealed the endocytic pathwayby which these lipoprotein particles were internalized

7. Postulatedthat … • The rapid internalization of receptor-bound LDL is strictly dependent on the localization of LDL receptors in coated pits. • If an LDL receptor failed to become localized within a coated pit, it would be unable to deliver its bound ligand to cellular lysosomes and thus would be unable to affect cholesterol（胆固醇） biosynthesis within the cell.

8. The LDL receptor • J.D. mutation At about this time, an LDL receptor with a different type of mutation was discovered. LDL receptors bearing this new defect bound normal amounts of radioactively labeled LDL, yet the receptor-bound lipoprotein failed to internalized and consequently was not delivered to cytoplasmic lysosomes for processing.

9. Structure of the LDL receptor Atransmembrane glycoprotein of 839 amino acids, with the 50 amino acids at the c-terminal end of the protein extending inward from the membrane as a cytoplasmic domain Its cytoplasmic domain was specifically bound by a likely subunit of an AP adaptor of the coated pits.

10. J.D. mutation • Analysis of the J.D. mutant receptor revealed that the protein contained a single amino acid substitution: a tyrosine(酪氨酸) residue normally located at position 807 was replaced by a cysteine(半胱氨酸). This single alteration in amino acid sequence obliterated the ability of the protein to become concentrated in coated pits.

11. J.D. mutant receptor (unable to become localized in a cell’s coatedpits)

12. Over the next few years, attention turned to the amino acid sequences of the cytoplasmic tails of other receptors that become localized in coated pits. Was there a common internalization signal? Internalization signals

13. NPXYsignal as in the LDL receptor YXXφ signalas in the transferrin（铁 转运蛋白） receptor Y — tyrosine X — any amino acid φ— an amino acid with a bulky hydrophobic side chain （疏水侧链） Two signals

14. The YXXφ sequence of the receptor binds to the μ subunit of the AP2 adaptors. AP2 adaptor complex binds to the clathrin coat by means of its β subunit.

15. μ subunit（gray）have tow hydrophobic pockets one that binds the tyr and the other that binds the bulky hydrophobic side chain of the internalization signal (purple). The interaction between the adaptor and internalization signal

16. Many Thanks