Figure 7. ANP-induced receptor down-regulation in HEK-293 cells expressing wild-type or mutant receptors.

Confluent HEK-293 cells expressing wild-type, G920A, D921A, Y923A or GDAY/AAAA mutant receptors in 6 cm² culture dishes were pretreated with unlabelled ANP (10 nM) for the indicated time periods. Cells were then washed with glycine acidic buffer (pH 3.8) to remove any surface-bound ¹²⁵I-ANP. After washing the cells four times with assay medium (2 ml each), cells were reincubated in 2 ml of fresh assay medium. The ¹²⁵I-ANP binding was assayed at the indicated time periods as described in the Materials and methods section. The specific ¹²⁵I-ANP binding was then determined and expressed as a percentage of the initial radioligand ¹²⁵I-ANP binding. The results represent the means±S.E.M. for four independent determinations with triplicate dishes in each experiment.