A key role for protein kinase a in homologous desensitization of the β2-adrenergic receptor pathway in S49 lymphoma cells

Abstract

We have used a [3H]forskolin binding assay to assess Gs-adenylyl cyclase interactions in intact wild-type (WT) and kin- S49 cells under conditions that desensitize the β2-adrenergic receptor (β2-AR) system. This assay provides a measurement of Gαs-adenylyl cyelase interaction that does not rely on the determination of second messenger accumulation or enzyme activity in broken cells. Kin- S49 cells lack protein kinase A (PKA) activity and provide a unique system in which to study the relative importance of this enzyme in β2-AR desensitization. Although both WT and kin- S49 cells display similar kinetics of cAMP accumulation and agonist-induced cell-surface β2-AR loss, we found that these cell types exhibited very different extents of desensitization of forskolin binding following agonist treatment. Specifically, 10 μM isoproterenol (37 °C, 30 min) induced the loss of 70% of [3H]forskolin binding sites in WT cells but only 30% in kin- cells. This loss of sites in WT cells displayed a t1/2 of ≈ 7 min, was agonist concentration-dependent (EC50 ≈ 60 nM), was not mimicked by 8-Br-cAMP, and could be blocked by the PKA inhibitor, H89. The difference between WT and kin- cells in agonist-induced desensitization of the β2-AR pathway was also noted in studies of cAMP accumulation in cells. In addition, preincubation of intact cells with isoproterenol did not inhibit guanine nucleotide-dependent [3H]forskolin binding in permeabilized cells. Overall, data obtained from [3H]forskolin binding assays demonstrate the involvement of PKA in the agonist-dependent uncoupling of β2-AR and Gs; thus we conclude that PKA plays an important role in the homologous desensitization of the β2-AR-Gs-adenylyl cyclase pathway in intact cells.