The human α1D-adrenergic receptor is a seven transmembrane-domain protein that mediates many of the physiological actions of adrenaline and noradrenaline and participates in the development of hypertension and benign prostatic hyperplasia. We recently reported that different phosphorylation patterns control α1D-adrenergic receptor desensitization. However, to our knowledge, there is no data regarding the role(s) of this receptor's specific phosphorylation residues in its subcellular localization and signaling. In order to address this issue, we mutated the identified phosphorylated residues located on the third intracellular loop and carboxyl tail. In this way, we experimentally confirmed α1D-AR phosphorylation sites and identified, in the carboxyl tail, two groups of residues in close proximity to each other, as well as two individual residues in the proximal (T442) and distal (S543) regions. Our results indicate that phosphorylation of the distal cluster (T507, S515, S516 and S518) favors α1D-AR localization at the plasma membrane, i. e., substitution of these residues for non-phosphorylatable amino acids results in the intracellular localization of the receptors, whereas phospho-mimetic substitution allows plasma membrane localization. Moreover, we found that T442 phosphorylation is necessary for agonist- and phorbol ester-induced receptor colocalization with β-arrestins. Additionally, we observed that substitution of intracellular loop 3 phosphorylation sites for non-phosphorylatable amino acids resulted in sustained ERK1/2 activation; additional mutations in the phosphorylated residues in the carboxyl tail did not alter this pattern. In contrast, mobilization of intracellular calcium and receptor internalization appear to be controlled by the phosphorylation of both third-intracellular-loop and carboxyl terminus-domain residues. In summary, our data indicate that a) both the phosphorylation sites present in the third intracellular loop and in the carboxyl terminus participate in triggering calcium signaling and in turning-off α1D-AR-induced ERK activation; b) phosphorylation of the distal cluster appears to play a role in receptor's plasma membrane localization; and c) T442 appears to play a critical role in receptor phosphorylation and receptor-β-arrestin colocalization.

中文翻译：

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羧基末端的不同磷酸化位点/簇调节α1D-肾上腺素能受体亚细胞的定位和信号传导。

人α1D-肾上腺素受体是七个跨膜结构域蛋白，可介导肾上腺素和去甲肾上腺素的许多生理作用，并参与高血压和良性前列腺增生的发展。我们最近报道了不同的​​磷酸化模式控制α1D-肾上腺素能受体脱敏。然而，据我们所知，没有关于该受体的特定磷酸化残基在其亚细胞定位和信号传导中的作用的数据。为了解决这个问题，我们突变了位于第三个细胞内环和羧基尾部的已鉴定的磷酸化残基。这样，我们通过实验确定了α1D-AR的磷酸化位点，并在羧基尾部确定了彼此接近的两组残基，以及在近端（T442）和远端（S543）区域中的两个单独的残基。我们的结果表明，远端簇（T507，S515，S516和S518）的磷酸化有利于质膜上的α1D-AR定位，即用这些残基替代不可磷酸化的氨基酸会导致受体在细胞内定位，而磷酸模拟取代允许质膜定位。此外，我们发现，T442磷酸化对于激动剂和佛波酯诱导的受体与β-arrestin的共定位是必要的。此外，我们观察到细胞内环3磷酸化位点被不可磷酸化的氨基酸取代会导致持续的ERK1 / 2激活。羧基尾部的磷酸化残基中的其他突变不会改变这种模式。相比之下，细胞内钙的动员和受体内在化似乎受第三细胞内环和羧基末端结构域残基的磷酸化的控制。总而言之，我们的数据表明：a）存在于第三细胞内环和羧基末端的磷酸化位点均参与触发钙信号传导并关闭α1D-AR诱导的ERK激活；b）远端簇的磷酸化似乎在受体的质膜定位中起作用；c）T442似乎在受体磷酸化和受体-β-arrestin共定位中起关键作用。我们的数据表明：a）存在于第三个细胞内环和羧基末端的磷酸化位点均参与触发钙信号传导并关闭α1D-AR诱导的ERK激活；b）远端簇的磷酸化似乎在受体的质膜定位中起作用；c）T442似乎在受体磷酸化和受体-β-arrestin共定位中起关键作用。我们的数据表明：a）存在于第三个细胞内环和羧基末端的磷酸化位点均参与触发钙信号传导并关闭α1D-AR诱导的ERK激活；b）远端簇的磷酸化似乎在受体的质膜定位中起作用；c）T442似乎在受体磷酸化和受体-β-arrestin共定位中起关键作用。