cAMP binding to the cell surface and its inhibition by GTPγS. (A) Scatchard analysis of cAMP binding in AX2 and HSB61 cells pulsed with cAMP for 5 or 7 hours, respectively. The [3H] cAMP binding was determined over a range of 700–19700 nM cAMP by incubation for 5 min at 0°C. A fitted line, Bmax and Kd are shown in each panel. (B) Inhibitory effect of GTPγS (black bar) on the binding of cAMP to their cognate receptors. Crude membranes were incubated with [3H] cAMP in the absence or presence of GTPγS. Values are indicated as the percentage of cAMP binding in treated normalized to the untreated membranes (white bar). In wild type strain cAMP binding dropped from 10.4 ± 0.3 nM to 5.26 ± 0.04 nM for GTPγS untreated and treated membranes, respectively. HSB61 strain displayed comparable absolute values: 16.23 ± 0.25 and 16.00 ± 0.3 nM for GTPγS untreated and treated membranes, respectively. Means of three independent experiments, each run in duplicate, are shown.
Arigoni et al. BMC Cell Biology 2005 6:43 doi:10.1186/1471-2121-6-43