Clathrin lattice assembly and adaptor recruitment in receptor-mediated endocytosis
Receptor-mediated endocytosis at the plasma membrane occurs via clathrin-coated pits. The clathrin coat is believed to integrate dual functions: (1) It serves as structural scaffolding, supporting the progressive invagination of underlying plasma membrane which culminates in the formation of a coated vesicle; and (2) It possesses a molecular adaptor function, ensuring the selective inclusion of transmembrane receptors and their ligands destined for an intracellular compartment. This study examines aspects of both functions. Evidence is presented for a mechanism by which clathrin assembly occurs at the plasma membrane by showing that in vitro translated α and β2 subunits bind independently to clathrin trimers and cages. This confirms that AP-2 is bivalent and is consistent with clathrin assembly occurring via a crosslinking mechanism, in which two clathrin protomers are linked by a single AP-2 complex. In particular, α subunit binds with high affinity to pure clathrin cages, and this binding is inhibited by intact AP-2 complexes, indicating specificity. Limited proteolysis reveals that both subunits are digested into core and appendage domains, consistent with their native tertiary structure. While the appendages am stable to further digestion, both core domains are labile relative to those derived from AP-2, consistent with the absence of interchain contacts present in heterotetrameric, AP-2. By deletion analysis, the N-terminal core of α contains the clathrin binding region and limited proteolysis of bound α reveals a 29 kDa stretch of residues which remains bound to cages and likely constitutes a clathrin binding subdomain. Upon agonist binding, the β2-adrenergic receptor is rapidly phosphorylated and recruits an arrestin molecule. Here we show that nonvisual arrestin isoforms bind clathrin lattices with high affinity and with a 3 mol arrestin/mol of clathrin stoichiometry in vitro. Together with the observation that in COS-1 cells, arrestin, receptor, and clathrin colocalize in an agonist-dependent and arrestin-dependent manner, this suggests that arrestins, by recruiting activated receptors to coated pits, fulfill a novel adaptor function. Limited proteolysis of cages reveals arrestin binding is lost upon clathrin terminal domain release, independently of light chains, indicating that the terminal domain binds arrestin. Finally, deletion mutagenesis, alanine scanning mutagenesis, and site-directed mutagenesis on a construct consisting of GST fused to clathrin terminal domain demonstrate that residues 89–100 of clathrin heavy chain are an important determinant for arrestin binding.
Pharmacology|Cellular biology|Molecular biology
Goodman, Oscar Baylin, "Clathrin lattice assembly and adaptor recruitment in receptor-mediated endocytosis" (1999). ProQuest ETD Collection - Thomas Jefferson University. AAI9921017.