Table 2. Quantification of the total and intracellular pool of wild-type and GDAY/AAAA mutant NPRA in intact and solubilized recombinant HEK-293 cells treated with and without trypsin.

HEK-293 cells expressing wild-type or GDAY/AAAA mutant receptors were washed with binding assay medium (DMEM containing 0.1% BSA) and cells were allowed to bind ¹²⁵I-ANP in control and trypsin-treated (0.025%) groups. In parallel experiments, the control and trypsin-treated cells were solubilized in a buffer containing 1% Triton X-100, 15% glycerol and protease inhibitors. The binding of ¹²⁵I-ANP in intact and solubilized cells was determined as described in the Materials and methods section. The percentage relative binding was determined from the specific ¹²⁵I-ANP binding parameters. The results represent the means±S.E.M. for four independent measurements.

	Wild-type NPRA		GDAY/AAAA NPRA
Treatment	Specific 125I-ANP binding (%)	Relative binding (%)	Specific 125I-ANP binding (%)	Relative binding (%)
Untreated	20.64±2.92	100	21.03±3.01	100
Trypsin-treated	1.48±0.16	7.17	1.76±0.14	5.99
Triton X-100-solubilized	25.73±2.81	100	23.92±2.54	100
Trypsin plus Triton X-100	6.28±0.30	24.40	4.46±0.28	18.68