We have used a [H]forskolin binding assay to assess G-adenylyl cyclase interactions in intact wild-type (WT) and kin S49 cells under conditions that desensitize the -adrenergic receptor (-AR) system. This assay provides a measurement of G-adenylyl cyclase 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 -AR desensitization. Although both WT and kin S49 cells display similar kinetics of cAMP accumulation and agonist-induced cell-surface -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 [H]forskolin binding sites in WT cells but only 30% in kin cells. This loss of sites in WT cells displayed a t of 7 min, was agonist concentration-dependent (EC 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 -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 [H]forskolin binding in permeabilized cells. Overall, data obtained from [H]forskolin binding assays demonstrate the involvement of PKA in the agonist-dependent uncoupling of -AR and G; thus we conclude that PKA plays an important role in the homologous desensitization of the -AR-G-adenylyl cyclase pathway in intact cells.

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