Conformational changes in the receptors for epidermal growth factor and asialoglycoproteins induced by the mildly acidic pH found in endocytic vesicles.
We have used a variety of methods, including lactoperoxidase-catalyzed iodination, proteolysis, and photolabel incorporation, to determine whether exposure to the acidic pH encountered during receptor-mediated endocytosis causes observable conformational changes in receptor proteins. Two receptor systems were chosen for this study: the asialoglycoprotein receptor and the epidermal growth factor (EGF) receptor. The purified asialoglycoprotein receptor protein was reconstituted into lipid membranes by spontaneous incorporation into phosphatidylcholine liposomes with the binding site facing outward. The EGF receptor was studied in living A-431 cells and was identified by immunoprecipitation using monoclonal antibodies. Lactoperoxidase-catalyzed iodination of both receptor systems, carried out with the external pH equal to 7.4 or 5.6, showed that the extent of receptor protein iodination was less at the lower pH even though lactoperoxidase has an acidic pH optimum. Using the nonspecific hydrophilic photolabeling agent [35S]N-(4-azido-3-nitrophenyl)-2-aminoethylsulfonic acid-taurine, we observed less incorporation into both the asialoglycoprotein receptor in liposomes and the EGF-receptor in A-431 cells when the external pH was reduced to 5.6. Also, using the enzyme papain, we have found that both receptors become resistant to proteolysis when the external pH is lowered from 7.0 to 5.6. These results suggest a conformational change in both of these receptors in which they become less exposed to the external aqueous environment at low pH. Such a conformational change may be responsible for the pH dependence of binding for both of these ligands. Also, this conformational change may serve to protect receptors from enzymatic degradation within endocytic or lysosomal compartments.
D037263:Asialoglycoprotein Receptor; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D009961:Orosomucoid / Q000031:analogs & derivatives; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism*
D037263:Asialoglycoprotein Receptor; D008081:Liposomes; D008565:Membrane Proteins; D009961:Orosomucoid; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D066246:ErbB Receptors