Role of the cyclic AMP-dependent protein kinase in homologous resensitization of the β1-adrenergic receptor
Role of the cyclic AMP-dependent protein kinase in homologous resensitization of the β1-adrenergic receptor
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DOI：
10.1074/jbc.m313652200
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发表时间：
2004-05-14
影响因子：
4.8
通讯作者：
Bahouth, SW
中科院分区：
文献类型：
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作者：
Gardner, LA;Delos Santos, NM;Bahouth, SW
生物学中的一个基本问题是G蛋白偶联受体中的各种基序如何参与这些分子协调的不同功能。在这里，我们描述了一个基本的作用，丝氨酸残基在位置312在第三个细胞内环的人β（1）-肾上腺素能受体（β（1）-AR）的激动剂内化受体的内吞再循环。在通过共聚焦显微镜监测的受体再循环实验中，激动剂内化野生型（WT）β（1）-AR再循环试验Ser（312）突变为丙氨酸（Ser（312）--> Ala β（1）-AR）或突变为磷酸丝氨酸模拟物天冬氨酸（Ser（312 -->）Asp β（1）-AR）导致与WT β（1）-AR几乎不可区分的β（1）-AR构建体。内化的Ser（312）--> Asp beta（1）-AR有效地再循环，t（0.5）为11 +/- 3 min，而内化的Ser（312）--> Ala beta（1）-AR不通过内体途径再循环或功能性再敏化。由于丝氨酸是环腺苷酸依赖性蛋白激酶（PKA）磷酸化的假定残基，我们研究了该激酶在内化β（1）-AR再循环中的作用。使用显性阴性PKA构建体在生化或遗传上抑制PKA，阻断了内化WT β（1）-AR的再循环。磷酸化研究表明，β（1）-AR被PKA部分磷酸化，PKA催化亚基对β（1）-AR的磷酸化仅发生在Ser（312）。我们的研究结果确定了一个新的信号模式，其中同源激活的激酶提供了一个可逆的修改，转向行程的内化受体回收和再敏化。因此，PKA介导的G蛋白偶联受体磷酸化可能导致基序依赖性脱敏或再敏化。
A fundamental question in biology is how the various motifs in G protein-coupled receptors participate in the divergent functions orchestrated by these molecules. Here we describe a fundamental role for a serine residue at position 312 in the third intracellular loop of the human beta(1)-adrenergic receptor (beta(1)-AR) in endocytic recycling of the agonist-internalized receptor. In receptor recycling experiments that were monitored by confocal microscopy, the agonist-internalized wild-type (WT) beta(1)-AR recycled with a t(0.5) of 14 +/- 3 min. Mutagenesis of Ser(312) to alanine (Ser(312) --> Ala beta(1)-AR) or to the phosphoserine mimic aspartic acid (Ser(312 -->) Asp beta(1)-AR) resulted in beta(1)-AR constructs that were pharmacologically indistinguishable from the WT beta(1)-AR. The internalized Ser(312) --> Asp beta(1)-AR recycled efficiently with a t(0.5) of 11 +/- 3 min, whereas the internalized Ser(312) --> Ala beta(1)-AR was not recycled or functionally resensitized through the endosomal pathway. Because this serine is a putative residue for phosphorylation by the cyclic AMP-dependent protein kinase (PKA), we examined the role of this kinase in recycling of the internalized beta(1)-AR. Inhibition of PKA biochemically or genetically using a dominant negative PKA construct blocked the recycling of the internalized WT beta(1)-AR. Phosphorylation studies revealed that the beta(1)-AR is partially phosphorylated by PKA and that phosphorylation of the beta(1)-AR by the catalytic subunit of PKA occurs exclusively at Ser(312). Our results identify a new signaling paradigm in which homologous activation of a kinase provides a reversible modification that shifts the itinerary of the internalized receptor toward recycling and resensitization. Therefore, PKA-mediated phosphorylation of G protein-coupled receptors might result in motif-dependent desensitization or resensitization.