Spurney R F
Division of Nephrology, Department of Medicine, Duke University and Durham Veterans Affairs Medical Centers, Durham, North Carolina 27710, USA.
J Biol Chem. 1998 Oct 23;273(43):28496-503. doi: 10.1074/jbc.273.43.28496.
To investigate the role of C-terminal hydroxyamino acids in desensitization of the receptor for thromboxane A2 (TxA2), we created a mutant TxA2 receptor (TP receptor) in which serines at positions 321, 322, and 328 were replaced with either alanine or glycine. Mutant and wild type receptors were expressed in a mesangial cell line, and clones expressing similar numbers of receptors were studied. Affinity and specificity of TxA2 binding to the mutant receptor were identical to wild type receptors. In contrast, TxA2-induced inositol trisphosphate generation by the mutant receptor was enhanced compared with the wild type. Prior treatment with the TxA2 agonist U46619 reduced subsequent U46619-induced increases in inositol trisphosphate generation by both receptors; however, the extent of desensitization was significantly reduced in the receptor mutant. Protein kinase C (PKC) inhibitors attenuated TxA2-induced desensitization of wild type receptors, but had little effect on TxA2-induced desensitization of mutant receptors. Pretreatment with the phorbol ester phorbol 12, 13-dybutyrate (PDBu) (100 nM) decreased subsequent responsiveness of wild type but not mutant TP receptors. -induced desensitization of wild type receptors was associated with enhanced phosphorylation of receptor proteins. This agonist-specific phosphorylation of the TP receptor was largely prevented by inhibitors of PKC. Treatment with 100 nM PDBu increased phosphorylation of both wild type and mutant TP receptors, but the extent of phosphorylation of the receptor mutant was reduced compared with the wild type. Increasing the concentration of PDBu from 100 nM to 1 microM PDBu reduced responsiveness of both mutant and wild type receptors without enhancing phosphorylation of either of the receptor proteins. These data suggest that 1) phosphorylation of C-terminal serines contributes to agonist-specific desensitization of the TP receptor, 2) PKC-induced desensitization of TP receptors is caused, in part, by phosphorylation of C-terminal serines, and 3) desensitization of TP receptors by PKC is complex and involves mechanisms that may not require direct phosphorylation of receptor proteins.
为了研究C末端羟基氨基酸在血栓素A2（TxA2）受体脱敏中的作用，我们构建了一种突变型TxA2受体（TP受体），其中321、322和328位的丝氨酸被丙氨酸或甘氨酸取代。突变型和野生型受体在系膜细胞系中表达，并对表达相似数量受体的克隆进行研究。TxA2与突变型受体结合的亲和力和特异性与野生型受体相同。相比之下，突变型受体诱导的肌醇三磷酸生成比野生型增强。用TxA2激动剂U46619预先处理可降低随后U46619诱导的两种受体的肌醇三磷酸生成增加；然而，受体突变体中的脱敏程度显著降低。蛋白激酶C（PKC）抑制剂可减弱TxA2诱导的野生型受体脱敏，但对TxA2诱导的突变型受体脱敏作用很小。用佛波酯佛波醇12,13 - 二丁酸酯（PDBu）（100 nM）预处理可降低野生型而非突变型TP受体随后的反应性。野生型受体的激动剂特异性脱敏与受体蛋白磷酸化增强有关。PKC抑制剂可很大程度上阻止TP受体这种激动剂特异性磷酸化。用100 nM PDBu处理可增加野生型和突变型TP受体的磷酸化，但与野生型相比，受体突变体的磷酸化程度降低。将PDBu浓度从100 nM增加到1 μM可降低突变型和野生型受体的反应性，而不会增强任何一种受体蛋白的磷酸化。这些数据表明：1）C末端丝氨酸的磷酸化有助于TP受体的激动剂特异性脱敏；2）PKC诱导的TP受体脱敏部分是由C末端丝氨酸的磷酸化引起的；3）PKC对TP受体的脱敏作用很复杂，涉及可能不需要受体蛋白直接磷酸化的机制。
