EVIDENCE FOR HAPTEN RECOGNITION IN RECEPTOR-MEDIATED INTRACELLULAR UPTAKE OF A HAPTEN-PROTEIN CONJUGATE BY MURINE MACROPHAGE

Fluorescein-derivatized bovine serum albumin (FITC-BSA) was used as an exogenous antigen and fluorescent probe to measure the kinetics of an tigen uptake into the endocytic pathway of murine macrophage, J774, us ing flow cytometry. Results revealed dependency of the rate of antigen uptake on epitope density (moles FITC/mole BSA) implicating a role fo r FITC in the endocytosis of the derivatized antigen. In addition, inh ibition of clathrin-coated pit formation in macrophage resulted in sig nificantly reduced uptake of differentially labeled FITC-BSA probes in dicating receptor-mediated endocytosis via clathrin-coated pits. Fluor esceinamine (I) was found to inhibit the endocytic uptake of FITC-BSA at 10(-6)M. Determination of fractional receptor occupancies in macrop hage upon binding different FITC-BSA probes and calculation of the cor responding association rates (k(on)) for these binding events yielded values of 4.2+/-0.2 x 10(6)/M/min for FITC(5)BSA and 1.9+/-0.1 x 10(7) /M/min for FITC(22)BSA, respectively, at 37 degrees C. The five-fold d ifference in the rates of binding and endocytosis between the two prob es was discussed on the basis of receptor cross-linking by a multivale nt ligand (FITC(22)BSA), in contrast to monovalent ligand binding, on the cell surface that would lead to more rapid and efficient internali zation of the FITC(22)BSA antigen. (C) 1997 Elsevier Science Ltd.