Phosphorylation of the angiotensin II (AT1A) receptor carboxyl terminus: a role in receptor endocytosis.
Phosphorylation of the angiotensin II (AT1A) receptor carboxyl terminus: a role in receptor endocytosis.
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DOI：
10.1210/mend.12.10.0179
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发表时间：
1998-10
影响因子：
--
通讯作者：
Walter G. Thomas;Thomas J. Motel;C. Kule;Vijay Karoor;Kenneth M. Baker
中科院分区：
文献类型：
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作者：
Walter G. Thomas;Thomas J. Motel;C. Kule;Vijay Karoor;Kenneth M. Baker
血管紧张素II I型受体(AT1)内吞作用的分子机制尚不清楚，尽管在AT1A型受体的羧基末端发现了一个重要的丝氨酸/苏氨酸富集区(Thr332Lys333Met334Ser335Thr336Leu337 Ser338)，提示可能与磷酸化有关。在这项研究中，我们检测了全长AT1a受体的磷酸化和内化，并将其与羧基末端截短和突变的受体进行了比较。表位标记的全长AT1a受体瞬时转染中国仓鼠卵巢(CHO)-K1细胞后，基础水平的磷酸化水平被血管紧张素II(Ang II)刺激显著增强。AT1a受体的磷酸化水平随着羧基末端的连续截断而逐渐降低，并被截断到Lys325，去掉了最后34个氨基酸，几乎完全抑制了Ang II刺激的32P掺入AT1a受体。为了研究受体磷酸化与内吞作用的相关性，我们构建了一个表位标记的突变体受体，用丙氨酸取代了先前被认为对受体内化有重要作用的羧基末端残基Thr332、Ser335、Thr336和Ser338。与野生型受体相比，该突变体明显减少了Ang II刺激的磷酸化。Ang II多肽拮抗剂Sar(1)Ile8-Ang II矛盾地导致野生型AT1a受体内化，也促进了它们的磷酸化，这一新观察进一步加强了这种相关性。为了将羧基末端的磷酸化与内吞作用直接联系起来，比较了野生型AT1a受体和Thr332-Ser338区突变受体的内化动力学。四个可能的磷酸化位点(Thr332、Ser335、Thr336和Ser338)被中性氨基酸[丙氨酸(A)]或酸性氨基酸[谷氨酸(E)和天冬氨酸(D)]取代，前者用于阻止磷酸化，后者用于复制磷酸化产生的酸性电荷。在中国仓鼠卵巢细胞中表达的野生型AT1a受体在Ang II刺激后迅速内化[T1/2 2.3min；最大内化水平(Ymax)78.2%]，携带单一酸性取代的突变受体(T332E，T1/2 2.7min，Ymax 76.3%；S335D，T1/2 2.4min，Ymax 76.7%；T336E，T1/2 2.5min，Ymax 78.2%；S338D，T1/2 2.6min，Ymax 78.4%)。虽然酸性氨基酸替换可能不像丙氨酸突变那样在结构上具有破坏性，但我们将该区域对负电荷的耐受解释为提示磷酸化可能允许最大限度的内化。4个氨基酸残基均被丙氨酸取代，产生内化动力学显著降低的受体(T332A/S335A/T336A/S338A，T1/2 10.1min，Ymax 47.9%)，而相应的四重酸性突变体(T332E/S335D/T336E/S338D，T1/2 6.4min，Ymax 53.4%)显著恢复内吞作用。S335和T336双突变为丙氨酸也降低了内吞的速度和程度(S335A/T336A，3.9min，Ymax 69.3%)，而类似的双酸突变体表现出类似野生型的内吞参数(S335D/T336E，T1/2 2.6min，Ymax 77.5%)。基于我们已经确定的Ang II诱导的磷酸化部位的酸性氨基酸替换对内化的明显挽救，我们得出结论，AT1a受体的最大内吞作用需要在羧基末端的这一富含丝氨酸/苏氨酸的片段内磷酸化。
The molecular mechanism of angiotensin II type I receptor (AT1) endocytosis is obscure, although the identification of an important serine/threonine rich region (Thr332Lys333Met334Ser335Thr336Leu337 Ser338) within the carboxyl terminus of the AT1A receptor subtype suggests that phosphorylation may be involved. In this study, we examined the phosphorylation and internalization of full-length AT1A receptors and compared this to receptors with truncations and mutations of the carboxyl terminus. Epitope-tagged full-length AT1A receptors, when transiently transfected in Chinese hamster ovary (CHO)-K1 cells, displayed a basal level of phosphorylation that was significantly enhanced by angiotensin II (Ang II) stimulation. Phosphorylation of AT1A receptors was progressively reduced by serial truncation of the carboxyl terminus, and truncation to Lys325, which removed the last 34 amino acids, almost completely inhibited Ang II-stimulated 32P incorporation into the AT1A receptor. To investigate the correlation between receptor phosphorylation and endocytosis, an epitope-tagged mutant receptor was produced, in which the carboxyl-terminal residues, Thr332, Ser335, Thr336, and Ser338, previously identified as important for receptor internalization, were substituted with alanine. Compared with the wild-type receptor, this mutant displayed a clear reduction in Ang II-stimulated phosphorylation. Such a correlation was further strengthened by the novel observation that the Ang II peptide antagonist, Sar(1)Ile8-Ang II, which paradoxically causes internalization of wild-type AT1A receptors, also promoted their phosphorylation. In an attempt to directly relate phosphorylation of the carboxyl terminus to endocytosis, the internalization kinetics of wild-type AT1A receptors and receptors mutated within the Thr332-Ser338 region were compared. The four putative phosphorylation sites (Thr332, Ser335, Thr336, and Ser338) were substituted with either neutral [alanine (A)] or acidic amino acids [glutamic acid (E) and aspartic acid (D)], the former to prevent phosphorylation and the latter to reproduce the acidic charge created by phosphorylation. Wild-type AT1A receptors, expressed in Chinese hamster ovary cells, rapidly internalized after Ang II stimulation [t1/2 2.3 min; maximal level of internalization (Ymax) 78.2%], as did mutant receptors carrying single acidic substitutions (T332E, t1/2 2.7 min, Ymax 76.3%; S335D, t1/2 2.4 min, Ymax 76.7%; T336E, t1/2 2.5 min, Ymax 78.2%; S338D, t1/2 2.6 min, Ymax 78.4%). While acidic amino acid substitutions may simply be not as structurally disruptive as alanine mutations, we interpret the tolerance of a negative charge in this region as suggestive that phosphorylation may permit maximal internalization. Substitution of all four residues to alanine produced a receptor with markedly reduced internalization kinetics (T332A/S335A/T336A/S338A, t1/2 10.1 min, Ymax 47.9%), while endocytosis was significantly rescued in the corresponding quadruple acidic mutant (T332E/S335D/T336E/S338D, t1/2 6.4 min, Ymax 53.4%). Double mutation of S335 and T336 to alanine also diminished the rate and extent of endocytosis (S335A/T336A, 3.9 min, Ymax 69.3%), while the analogous double acidic mutant displayed wild type-like endocytotic parameters (S335D/T336E, t1/2 2.6 min, Ymax 77.5%). Based on the apparent rescue of internalization by acidic amino acid substitutions in a region that we have identified as a site of Ang II-induced phosphorylation, we conclude that maximal endocytosis of the AT1A receptor requires phosphorylation within this serine/threonine-rich segment of the carboxyl terminus.