Fig. 9. Detection of agonist-mediated internalization of ß-subtypes by cell surface biotinylation. Arr2-BHK-hß₁ and -hß₂ cells were induced with zinc sulfate for 24 hours, incubated with or without 1 µM ISO for 15 minutes, and subjected to cell surface biotinylation for 30 minutes at 4°C, or biotinylated first and then exposed to agonist. The cells were lysed, extracted with detergent and clarified by centrifugation. The soluble receptors were immunoprecipitated with antibodies to ß₁AR or ß₂AR C-tail preabsorbed to protein A-agarose. The bound receptors were subjected to SDS-PAGE and blotting with HRP-streptavidin followed by chemiluminescence detection and densitometry. In addition, portions of the lysates were assayed for ¹²⁵ICYP binding, and separately, intact cells were assayed for [³H]CGP-12177 binding, to quantify the amounts of total and internalized receptors. (A) Blots of biotin-labeled ß₁AR (left) and ß₂AR (right) from control cells (C), cells exposed to ISO and biotinylated (I/B) and cells biotinylated and treated with ISO (B/I). Equal amounts of receptor (10 fmol of total ßAR) were loaded on the gel. (B) Summary of the quantification of cell surface receptor internalization by [³H]CGP-12177 binding and biotinylation by densitometry). Values are expressed as percentage of control and represent the means±s.e.m. of three separate experiments, each assayed in triplicate.