Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.
Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated alpha-adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by alpha-adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.
Chang
M P
MP
Department of Pharmacy, School of Pharmacy, University of California, San Francisco 94143-0446.
Mallet
W G
WG
Mostov
K E
KE
Brodsky
F M
FM
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D037263:Asialoglycoprotein Receptor; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000378:metabolism*; D012635:Secretory Component / Q000378:metabolism*
D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D037263:Asialoglycoprotein Receptor; D002966:Clathrin; D011506:Proteins; D011971:Receptors, Immunologic; D012635:Secretory Component