Agonist-selective mechanisms of μ-opioid receptor desensitization in human embryonic kidney 293 cells
Agonist-selective mechanisms of μ-opioid receptor desensitization in human embryonic kidney 293 cells
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DOI：
10.1124/mol.106.022376
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发表时间：
2006-08-01
影响因子：
3.6
通讯作者：
Henderson, Graeme
中科院分区：
文献类型：
--
作者：
Johnson, Elizabeth A.;Oldfield, Sue;Henderson, Graeme
研究了两种阿片激动剂[D-丙氨酸(2)，N-Me-苯丙氨酸(4)，甘氨酸(5)-醇]脑啡肽(DAMGO)和吗啡对表达大鼠MOR1和G蛋白偶联内向整流钾通道(GIRK)亚基的人胚胎肾(HEK)293细胞诱导MOR磷酸化、脱敏和内化的能力。DAMGO和吗啡均激活GIRK电流，但对DAMGO的最大反应大于吗啡，表明吗啡是一种部分激动剂。对DAMGO和吗啡的反应在任何一种药物存在下都会迅速脱敏。显性负性突变体G蛋白偶联受体激酶2(GRK2)GRK2-K220R的表达可显著减弱DAMGO对MOR1的脱敏作用，但对吗啡诱导的MOR1脱敏无明显影响。相反，蛋白激酶C(PKC)抑制肽PKC(19-31)或星形孢子素均可降低吗啡对MOR1的脱敏作用，但不能降低DAMGO的脱敏作用。吗啡和DAMGO可增强MOR1的基础磷酸化。PKC抑制剂双吲哚马来酰亚胺1(GF109203X)在基础条件下和吗啡存在时抑制MOR1的磷酸化，但不抑制DAMGO诱导的磷酸化。DAMGO诱导Arrestin-2转位至质膜，并使MOR1大量内化，而吗啡不诱导Arrestin-2转位，仅诱导极少量MOR1内化。因此，DAMGO和吗啡均可诱导HEK293细胞中MOR1信号的脱敏，但分子机制不同；DAMGO诱导的脱敏依赖于GRK2，而吗啡诱导的脱敏部分依赖于PKC。被DAMGO激活脱敏的MORs很容易被Arrestin依赖的机制内化，而被吗啡脱敏的MORs则不能。这些数据表明，阿片类激动剂诱导不同构象的MOR，这些构象对不同的脱敏和内化过程敏感。
The ability of two opioid agonists, [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkephalin ( DAMGO) and morphine, to induce mu-opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO-induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2-dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different desensitizing and internalization processes.