Stoichiometric interaction of the epidermal growth factor receptor with the clathrin-associated protein complex AP-2.
Plasma membrane clathrin-associated protein complexes (AP-2) have been shown to co-immunoprecipitate with the epidermal growth factor (EGF) receptor (Sorkin A., and Carpenter, G. (1993) Science 261, 612-615). Hence, we analyzed the stoichiometry of the EGF receptor interaction with AP-2 using a new antibody that efficiently immunoprecipitates native AP-2. EGF receptor AP-2 complexes were isolated from 35S-labeled cells treated with EGF by EGF receptor affinity chromatography followed by precipitation with the antibody to AP-2. Quantitation of the relative molar concentrations of the proteins found in the complex revealed that 1 mol of AP-2 was associated with approximately 1.1 mol of EGF receptor. No other proteins were present in significant molar concentrations relative to AP-2, indicating that other proteins are not stoichiometrically involved in the interaction of EGF receptors and AP-2 in vivo. Co-immunoprecipitation experiments in cells expressing a mutant EGF receptor demonstrated that the cytoplasmic carboxyl-terminal 214 residues of the EGF receptor are essential for interaction with AP-2.
Sorkin
A
A
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
McKinsey
T
T
Shih
W
W
Kirchhausen
T
T
Carpenter
G
G
D016475:3T3 Cells; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001483:Base Sequence; D002966:Clathrin / Q000378:metabolism*; D017931:DNA Primers; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism*; D010750:Phosphoproteins / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats
D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D002966:Clathrin; D017931:DNA Primers; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D066246:ErbB Receptors