The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation
The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation
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DOI：
10.1016/j.bbamcr.2013.06.013
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发表时间：
2013-10-01
影响因子：
5.1
通讯作者：
D'Ocon, P.
中科院分区：
文献类型：
--
作者：
Perez-Aso, M.;Segura, V.;D'Ocon, P.
我们分析了HEK 293细胞中三种α 1-肾上腺素受体（α 1-AR）亚型激活MAP激酶ERIC 1和2（ERK 1/2）的动力学和空间模式，以及两种不同途径对ERK 1/2磷酸化的贡献：蛋白激酶C（PKC）依赖性ERK 1/2激活和内化依赖性ERK 1/2激活。使用PKC抑制剂Ro 31-8425、受体内化抑制剂伴刀豆球蛋白A和靶向β-arrestin 2的siRNA，通过蛋白质印迹确定苯丙氨酸诱导ERIC磷酸化的不同途径。使用CypHer 5技术和VSV-G表位标记的受体研究了受体内化特性。苯丙氨酸对α（1A）-和α（1B）-AR的激活引起了ERK 1/2的快速磷酸化，该磷酸化被导向细胞核并被Ro 31-8425抑制。伴随着苯肾上腺素诱导的受体内化，α（1A）-AR，而不是α（1B）-AR，产生了维持和PKC非依赖性ERK磷酸化，这是限制在胞质溶胶和抑制蛋白2敲低或刀豆球蛋白A处理抑制。α（1D）-AR显示组成性ERIC磷酸化，与哌唑嗪或选择性am拮抗剂BMY 7378孵育可降低ERIC磷酸化。在苯肾上腺素激活后，α（1D）-AR引起快速、短暂的ERK 1/2磷酸化，其仅限于胞质溶胶，不受Ro 31-8425抑制。通过CypHer 5技术未观察到α（1D）-AR亚型的内化。三种α（1）-AR亚型呈现不同的受体内化时空模式，并且仅α（1A）-AR刺激转化为晚期持续的ERK 1/2磷酸化，其限于胞质溶胶并且依赖于β-抑制蛋白2介导的内化。(C)2013爱思唯尔有限公司版权所有。
We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERIC 1 and 2 (ERK1/2) by the three alpha(1)-adrenoceptor (alpha(1)-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERIC phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting beta-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of alpha(1A)- and alpha(1B)-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization alpha(1A)-AR, but not alpha(1B)-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by p-arrestin 2 knockdown or concanavalin A treatment. alpha(1D)-AR displayed constitutive ERIC phosphorylation, which was reduced by incubation with prazosin or the selective am antagonist BMY7378. Following activation by phenylephrine, alpha(1D)-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the alpha(1D)-AR subtype was not observed via CypHer5 technology. The three alpha(1)-AR subtypes present different spatio-temporal patterns of receptor internalization, and only alpha(1A)-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on beta-arrestin 2 mediated internalization. (C) 2013 Elsevier B.V. All rights reserved.