Oligomerization of the α1a- and α1b-adrenergic receptor subtypes -: Potential implications in receptor internalization
Oligomerization of the α1a- and α1b-adrenergic receptor subtypes -: Potential implications in receptor internalization
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DOI：
10.1074/jbc.m306085200
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发表时间：
2003-10-10
影响因子：
4.8
通讯作者：
Cotecchia, S
中科院分区：
文献类型：
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作者：
Stanasila, L;Perez, JB;Cotecchia, S
我们结合生物物理、生化和药理学方法来研究α(1a)和α(1b)肾上腺素能受体(AR)亚型形成同种和异种寡聚体的能力。标记了不同表位(血凝素和Myc)或荧光蛋白(青色和绿色荧光蛋白)的受体在HEK-293细胞中以单独或不同组合的形式瞬时表达。荧光共振能量转移测量提供了证据，α(1a)-AR和α(1b)-AR都可以形成均一低聚物，转移效率接近0.10。在α(1b)-AR亚型和α(1a)-AR亚型之间也可以观察到异源寡聚体，但在α(1b)-AR和β(2)-AR、NK1速激肽或CCR5趋化因子受体之间则没有。α(1b)-AR的齐聚不需要其C-尾、两个糖蛋白基序或其N末端的N-连接糖基化位点的完整性。相反，发现螺旋I和螺旋VII在α(1b)-AR同源寡聚中起作用。受体寡聚不受激动剂肾上腺素或反向激动剂哌唑嗪的影响。一个结构性活性(A293E)和一个信号缺陷(R143E)突变体表现出与野生型α(1b)-AR相似的寡聚特征。共聚焦成像显示，α(1)-AR亚型的寡聚化与它们在接触激动剂时共内化的能力相关。α(1a)选择性激动剂羟甲唑啉可诱导α(1a)-和α(1b)-AR共内化，而α(1b)-AR不能与NK1速激肽或CCR5趋化因子受体共内化。因此，寡聚可能代表了调节由α(1a)-和α(1b)-AR亚型介导的生理反应的另一种机制。
We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the alpha(1a)- and alpha(1b)-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the alpha(1a)- and alpha(1b)-AR can form homo-oligomers with similar transfer efficiency of similar to0.10. Hetero-oligomers could also be observed between the alpha(1b)- and the alpha(1a)-AR subtypes but not between the alpha(1b)-AR and the beta(2)-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the alpha(1b)-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the alpha(1b)-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type alpha(1b)-AR. Confocal imaging revealed that oligomerization of the alpha(1)-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The alpha(1a)-selective agonist oxymetazoline induced the co-internalization of the alpha(1a)- and alpha(1b)-AR, whereas the alpha(1b)-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the alpha(1a)- and alpha(1b)-AR subtypes.