A key role for protein kinase A in homologous desensitization of the beta(2)-adrenergic receptor pathway in S49 lymphoma cells

被引:35
|
作者
Post, SR [1 ]
AguilaBuhain, O [1 ]
Insel, PA [1 ]
机构
[1] UNIV CALIF SAN DIEGO, DEPT PHARMACOL, LA JOLLA, CA 92093 USA
关键词
D O I
10.1074/jbc.271.2.895
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We have used a [H-3]forskolin binding assay to assess G(s)-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 mu M isoproterenol (37 degrees C, 30 min) induced the loss of 70% of [H-3]forskolin binding sites in WT cells but only 30% in kin(-) cells, This loss of sites in WT cells displayed a t(1/2) of approximate to 7 min, was agonist concentration-dependent (EC(50) approximate to 60 nM), was not mimicked by 8-Br-cAMP, and could be blocked by the PHA 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 [H-3]forskolin binding in permeabilized cells, Overall, data obtained from [H-3]forskolin binding assays demonstrate the involvement of PKA in the agonist-dependent uncoupling of beta(2)-AR and G(s); thus we conclude that PKA plays an important role in the homologous desensitization of the beta(2)-AR-G(s)-adenylyl cyclase pathway in intact cells.
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收藏
页码:895 / 900
页数:6
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