FITC-poly-D-lysine conjugates as fluorescent probes to quantify hapten-specific macrophage receptor binding and uptake kinetics.
A series of fluorescein derivatized poly-D-lysine (FITC-PDL) probes were used to elucidate the role of fluorescein in receptor binding of fluorescein-conjugated macromolecules to J774 murine macrophages. Poly-D-lysine served to eliminate receptor recognition of the carrier due to the biologically inert nature of the D-isomer. This concept enabled the focused investigation of the role played by fluorescein in receptor recognition, binding and internalization. Results revealed dependency of cellular uptake on polymer concentration, hapten density and accessibility. The results differed from those previously obtained with FITC-BSA in that saturating fluorescein densities on the poly-D-lysine polymer resulted in diminished rates of uptake by macrophages. Receptor-mediated endocytosis via clathrin-coated pits was concluded based on results that showed inhibition of FITC-PDL uptake by intracellular K+ depletion but not by the macropinocytosis inhibitor, amiloride. Further, FITC-PDL was found to inhibit the endocytic uptake of FITC-BSA suggesting competition between the two probes at the level of a macrophage receptor. Association rates (kon) for binding to the macrophage surface were measured for the various FITC-PDL probes based on fractional receptor occupancies. Results are discussed on the basis of receptor recognition of fluorescein in J774 macrophages and the requirements for this recognition which include appropriate spacing and accessibility of the hapten moieties to facilitate receptor crosslinking.
Cherukuri
A
A
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana 61801, USA.
Frye
J
J
French
T
T
Durack
G
G
Voss
E W
EW
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D006241:Haptens / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008264:Macrophages / Q000166:cytology*; D051379:Mice; D008856:Microscopy, Fluorescence; D010873:Pinocytosis / Q000187:drug effects; D011188:Potassium / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012710:Serum Albumin, Bovine
D005456:Fluorescent Dyes; D006241:Haptens; D008024:Ligands; D011956:Receptors, Cell Surface; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; D000584:Amiloride; D016650:Fluorescein-5-isothiocyanate; D011188:Potassium