A Key Role for Protein Kinase A in Homologous Desensitization of the β2-Adrenergic Receptor Pathway in S49 Lymphoma Cells

Steven R Post,Olga Aguila-Buhain,Paul A Insel

doi:10.1074/jbc.271.2.895

Steven R Post, Olga Aguila-Buhain + Show 1 more

Open Access

https://doi.org/10.1074/jbc.271.2.895

Copy DOI

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 beta 2-adrenergic receptor (beta 2-AR) system. This assay provides a measurement of G alpha s-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 beta 2-AR desensitization. Although both WT and kin- S49 cells display similar kinetics of cAMP accumulation and agonist-induced cell-surface beta 2-AR loss, we found that these cell types exhibited very different extents of desensitization of forskolin binding following agonist treatment. Specifically, 10 microM isoproterenol (37 degrees 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 approximately 7 min, was agonist concentration-dependent (EC50 approximately 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 beta 2-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 beta 2-AR and Gs; thus we conclude that PKA plays an important role in the homologous desensitization of the beta 2-AR-Gs-adenylyl cyclase pathway in intact cells.

Highlights

In contrast to the effect of ␤-ARK, protein kinase A (PKA)-mediated ␤2-AR phosphorylation is most often associated with heterologous desensitization (that which is agonist nonspecific and 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) [11,12,13,14]
In contrast to the effect of ␤-ARK, protein kinase A (PKA)-mediated ␤2-AR phosphorylation is most often associated with heterologous desensitization, but is generally thought to be of little importance in homologous desensitization [11,12,13,14]
When WT cells were treated with the PKA inhibitor H89 (3 ␮M) the extent of isoproterenol-induced desensitization of [3H]forskolin binding was similar to that observed for kinϪ cells (Table I)

Summary

Introduction

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 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) [11,12,13,14]. These results indicate that the different extents of isoproterenol-induced desensitization of [3H]forskolin binding observed in WT and kinϪ cells results from cAMP-dependent activation of PKA.

Results

Conclusion