β2-adrenergic receptor lacking the cyclic AMP-dependent protein kinase consensus sites fully activates extracellular signal-regulated kinase 1/2 in human embryonic kidney 293 cells:: Lack of evidence for Gs/Gi switching.
β2-adrenergic receptor lacking the cyclic AMP-dependent protein kinase consensus sites fully activates extracellular signal-regulated kinase 1/2 in human embryonic kidney 293 cells:: Lack of evidence for Gs/Gi switching.
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DOI：
10.1124/mol.62.5.1094
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发表时间：
2002-11-01
影响因子：
3.6
通讯作者：
Clark, RB
中科院分区：
文献类型：
--
作者：
Friedman, J;Babu, B;Clark, RB
刺激人胚肾（HEK）293细胞中的β（2）-肾上腺素能受体（β（2）AR）导致细胞外信号调节激酶（ERK）1/2的瞬时激活。这种激活的机制之一是PKA介导的β 2 AR磷酸化，将受体偶联从G（s）转换为G（i），并触发受体的内化。为了检验这些现象，我们表征了内源性β 2 AR对HEK 293细胞中ERK 1/2的激动剂激活，以及稳定过表达野生型β 2 AR或缺乏环AMP依赖性蛋白激酶（PKA）共有磷酸化位点（S261 A、S262 A和S345 A、S346 A）的取代突变型β 2 AR（PKA（-））的HEK 293细胞中ERK 1/2的激动剂激活。作为基线，我们确定了肾上腺素刺激HEK 293细胞中的内源性β（2）AR（20-30 fmol/mg）引起ERK 1/2的快速和短暂激活，EC 50为5至6 nM。相比之下，肾上腺素刺激稳定过表达WT β（2）AR和PKA（-）的细胞中ERK 1/2的效力（2-4 pmol β（2）AR/mg）相对于HEK 293细胞增加超过100倍，EC 50值为20至60 pM。PKA（-）和WT beta（2）AR中ERK 1/2激活的EC 50相对于HEK 293中几乎相同的100倍偏移，表明PKA（-）完全能够激活ERK 1/2。我们还发现，在不引起内化的肾上腺素浓度下，ERK 1/2在过表达细胞系中的活化最大（即，内化EC 50为75 nM）。百日咳毒素预处理仅对HEK 293细胞中的ERK 1/2的肾上腺素活化产生弱抑制（7-16%），而对PKA（-）细胞中无抑制。最后，我们发现Src家族激酶抑制剂4-氨基-5-（4-氯苯基）-7-（叔丁基）吡唑并[3，4-d]嘧啶（10 μ M）在两种细胞系中均引起对肾上腺素或毛喉素激活ERK 1/2的>90%抑制。我们的研究结果表明，ERK 1/2的β（2）AR激活的主导机制不需要β（2）AR的PKA磷酸化、受体内化或从G（s）激活转换为G（i），但显然需要PKA下游的Src家族成员的激活。
Stimulation of the beta(2)-adrenergic receptor (beta(2)AR) in human embryonic kidney (HEK) 293 cells causes a transient activation of Extracellular Signal-Regulated Kinase (ERK) 1/2. One of the mechanisms proposed for this activation is a PKA-mediated phosphorylation of the beta(2)AR that switches receptor coupling from G(s) to G(i) and triggers internalization of the receptor. To examine these phenomena, we characterized agonist activation of ERK1/2 in HEK293 cells by the endogenous beta(2)AR and in HEK293 cells stably overexpressing either the wild-type beta(2)AR or a substitution mutant beta(2)AR (PKA(-)) that lacks the cyclic AMP-dependent protein kinase (PKA) consensus phosphorylation sites (S261A, S262A and S345A, S346A). As the baseline, we established that epinephrine stimulation of the endogenous beta(2)AR in HEK293 cells (20-30 fmol/mg) caused a rapid and transient activation of ERK1/2 with an EC50 of 5 to 6 nM. In contrast, the potency of epinephrine stimulation of ERK1/2 in cells stably overexpressing WTbeta(2)AR and PKA(-) (2-4 pmol of beta(2)AR/mg) was increased by over 100-fold relative to HEK293 cells, the EC50 values being 20 to 60 pM. The nearly identical 100-fold shift in EC50 for ERK1/2 activation in the PKA(-) and WTbeta(2)AR relative to that in the HEK293 showed that the PKA(-) are fully capable of activating ERK1/2. We also found maximal activation of ERK1/2 in the overexpressing cell lines at concentrations of epinephrine that cause no internalization (i.e., the EC50 for internalization was 75 nM). Pertussis toxin pretreatment caused only a weak inhibition of epinephrine activation of ERK1/2 in the HEK293 (7-16%) and no inhibition in the PKA(-) cells. Finally we found that the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-( t-butyl)pyrazolo[3,4-d]pyrimidine (10 muM) caused a >90% inhibition of epinephrine or forskolin activation of ERK1/2 in both cell lines. Our results indicate that the dominant mechanism of beta(2)AR activation of ERK1/2 does not require PKA phosphorylation of the beta(2)AR, receptor internalization or switching from activation of G(s) to G(i) but clearly requires activation of a Src family member that may be downstream of PKA.