Figure 5

Desensitization and Resensitization of β ₂ AR in wild-type HEK293 cells and cells in which AKAP12 or AKAP5 were knocked-down. A, cyclic β₂AR-mediated cyclic AMP accumulation in HEK cells that were desensitized by 30 min treatment with Iso and then washed free of agonist for 60 min (W60) to permit resensitization was examined. Agonist-stimulated (Iso,10 μM) cyclic AMP accumulation was measured in untreated cells, in cells following a 30-min prior stimulation with Iso, and in cells treated with Iso for 30 min, washed free of agonist, incubated for 60 min and then assayed (W60). HEK cells were pretreated with either a control, scrambled sequence siRNA (Control) or with siRNA to AKAP12 or AKAP5 to knock down (KD) the expression of endogenous AKAP12 or AKAP5. Cells were challenged with a β₂-adrenergic agonist (Iso,10 μM) for 30 min to provoke agonist-induced desensitization and internalization of β₂AR. The recovery from agonist-induced desensitization, termed resensitization, was measured in cells after a 30-min challenge with Iso, and in Iso-treated cells that were washed free of agonist for 60 min (W60). Isoproterenol-stimulated cyclic AMP accumulation was measured in these cells and is reported as picomol of cyclic AMP accumulated per 10⁵ cells. The results, displayed as mean ± S.E., are derived from at least three separate experiments performed with as many separate cultures of A431 cells. *, p ≤ 0.01 for the difference from the amount of cyclic AMP accumulation measured in the control cells at 60 min following a washout of the agonist (W60).