Tang H, Guo D F, Porter J P, Wanaka Y, Inagami T
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tenn 37232, USA.
Circ Res. 1998 Mar 23;82(5):523-31. doi: 10.1161/01.res.82.5.523.
To investigate mechanisms underlying the agonist-induced desensitization of the type 1A angiotensin II receptor (AT1A-R), we have stably expressed in Chinese hamster ovary (CHO) cells the wild-type receptor and truncated mutants lacking varying lengths of the cytoplasmic tail. Assay of inositol 1,4,5-trisphosphate (IP3) formation in response to agonist demonstrated that the truncated mutants T318, T328, and T348 lacking the last 42, 32, or 12 amino acid residues, respectively, couple with Gq protein with an efficiency similar to that of full-length receptors, whereas coupling of Gq protein was abolished in the T310 truncated mutant devoid of the carboxyl-terminal 50 amino acids. Exposure of CHO/AT1A-R cells expressing the wild-type AT1A-R to angiotensin II resulted in rapid and dose-dependent homologous desensitization of receptor-mediated IP3 formation, which was independent of the receptor internalization. Mastoparan, an activator of G protein-coupled receptor kinase (GRK), induced desensitization of the AT1A-R. The agonist-induced desensitization of the receptor was largely prevented by heparin, a potent inhibitor of GRK, whereas it was only partially attenuated by a protein kinase C (PKC)-specific inhibitor. The homologous or heterologous desensitization of the receptor was greatly impaired in the truncated mutants T318 and T328, lacking the Ser/Thr-rich (13 or 12 Ser/Thr residues) cytoplasmic tail of the AT1A-R. Deletion of the last two Ser residues, including one PKC consensus site in the receptor tail, prevented only phorbol 12-myristate 13-acetate-induced desensitization by 30%. Moreover, we found an agonist-induced translocation of a heparin-sensitive kinase activity. The angiotensin II-stimulated heparin-sensitive kinase could phosphorylate a thioredoxin fusion protein containing the entire AT1A-R cytoplasmic tail (N295 to E359), which lacks consensus phosphorylation sites for GRK1, GRK2, and GRK3. The heparin-sensitive kinase may not be GRK2, GRK3, or GRK6 expressed in CHO/AT1A-R cells, since angiotensin II did not induce translocation of these receptor kinases. Potential Ser/Thr phosphorylation sites located between S328 and S347 in the cytoplasmic tail of AT1A-R seem to play a critical role in the heterologous and homologous desensitization of the receptor. A heparin-sensitive kinase other than GRK2, GRK3, or GRK6 may be involved in the agonist-induced homologous desensitization of the AT1A-R.
为了研究1A型血管紧张素II受体（AT1A-R）激动剂诱导脱敏的潜在机制，我们在中华仓鼠卵巢（CHO）细胞中稳定表达了野生型受体和缺少不同长度细胞质尾的截短突变体。检测激动剂刺激后肌醇1,4,5-三磷酸（IP3）的生成，结果表明，分别缺失最后42、32或12个氨基酸残基的截短突变体T318、T328和T348与Gq蛋白的偶联效率与全长受体相似，而缺失羧基末端50个氨基酸的T310截短突变体则丧失了与Gq蛋白的偶联能力。表达野生型AT1A-R的CHO/AT1A-R细胞暴露于血管紧张素II后，受体介导的IP3生成出现快速且剂量依赖性的同源脱敏，这与受体内化无关。马斯托帕罗是一种G蛋白偶联受体激酶（GRK）激活剂，可诱导AT1A-R脱敏。GRK的强效抑制剂肝素可在很大程度上阻止激动剂诱导的受体脱敏，而蛋白激酶C（PKC）特异性抑制剂只能部分减弱这种脱敏作用。在缺少AT1A-R富含丝氨酸/苏氨酸（13或12个丝氨酸/苏氨酸残基）细胞质尾的截短突变体T318和T328中，受体的同源或异源脱敏作用受到极大损害。缺失受体尾中包括一个PKC共有位点的最后两个丝氨酸残基，仅使佛波醇12-肉豆蔻酸酯13-乙酸酯诱导的脱敏作用降低30%。此外，我们发现了一种激动剂诱导的肝素敏感激酶活性的转位。血管紧张素II刺激的肝素敏感激酶可磷酸化一种包含整个AT1A-R细胞质尾（N295至E359）的硫氧还蛋白融合蛋白，该细胞质尾缺乏GRK1、GRK2和GRK3的共有磷酸化位点。肝素敏感激酶可能不是CHO/AT1A-R细胞中表达的GRK2、GRK3或GRK6，因为血管紧张素II不会诱导这些受体激酶的转位。AT1A-R细胞质尾中位于S328和S347之间的潜在丝氨酸/苏氨酸磷酸化位点似乎在受体的异源和同源脱敏中起关键作用。一种不同于GRK2、GRK3或GRK6的肝素敏感激酶可能参与了激动剂诱导的AT1A-R同源脱敏。
