Desensitization of receptor-mediated cellular responses is a complex process which can involve multiple pathways including: (a) receptor uncoupling from G protein, (b) sequestration of receptors away from the plasma membrane, and (c) downregulation of signaling proteins (1-4). It has been demonstrated that uncoupling of receptor and G protein occurs rapidly (within 10 min) in response to receptor activation and appears to involve receptor phosphorylation (5). In the case of the ␤ 2 -adrenergic receptor (␤ 2 -AR) 1 system, agonist-occupied receptors activate the stimulatory G protein, G s , which in turn promotes the production of cAMP by adenylyl cyclase. Increased cAMP levels activate the cAMP-dependent protein kinase (PKA) resulting in the phosphorylation of several proteins, including the ␤ 2 -AR and other receptors. In addition, agonist-occupied ␤ 2 -AR are phosphorylated by one or more isoforms of the ␤-adrenergic receptor kinase (␤-ARK, also termed G protein receptor kinase; GRK) resulting in the uncoupling of receptor and G s (6 -10). Therefore, both PKA and ␤-ARK can contribute to desensitization of the ␤ 2 -AR system. In contrast to the effect of ␤-ARK, PKA-mediated ␤ 2 -AR phosphorylation is most often associated with heterologous desensitization (that which is agonist nonspecific and therefore not dependent on ␤ 2 -AR occupancy), but is generally thought to be of little importance in homologous desensitization (agonistspecific and dependent on ␤ 2 -AR occupancy) (11-14). Therefore, while the ␤ 2 -AR can be phosphorylated by both PKA and ␤-ARK, most recent work has focused on the importance of ␤-ARK and related proteins (reviewed in Refs. 6 and 15-17) as mediators of homologous desensitization.The kin Ϫ S49 murine lymphoma cell lacks PKA activity and thus provides a unique system in which to study the relative importance of PKA in mediating various cellular responses. Importantly, both WT and kin Ϫ S49 cells express only the ␤ 2 -AR isoform and display similar profiles of ␤-AR agonistinduced desensitization of cAMP production and cell-surface ␤ 2 -AR loss (14, 18). The ability of the cell-permeable cAMP analog, 8-Br-cAMP, to mimic the effect of epinephrine in decreasing ␤-AR agonist-induced adenylyl cyclase activity in membranes prepared from treated cells suggest that the effect of PKA activation is not dependent on the presence of agonist (11). These results suggest that PKA is not involved in homologous desensitization of the ␤ 2 -AR.In the current studies, we have used a [ 3 H]forskolin binding assay to define more precisely the role of PKA in desensitizing the ␤ 2 -AR system in intact S49 cells. Since high-affinity forskolin interaction with adenylyl cyclase requires the activation of G␣ s , the amount of [ 3 H]forskolin associated with cells can be used as an index of G␣ s -adenylyl cyclase interaction (19 -21). This assay, therefore, both provides a direct means of assessing hormone-stimulated G␣ s -adenylyl cyclase interaction in intact cells that is independent of second messenger accumulation...