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首页> 外文期刊>Biochemistry >Constitutive activation of the mu opioid receptor by mutation of D3.49(164), but not D3.32(147): D3.49(164) is critical for stabilization of the inactive form of the receptor and for its expression.
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Constitutive activation of the mu opioid receptor by mutation of D3.49(164), but not D3.32(147): D3.49(164) is critical for stabilization of the inactive form of the receptor and for its expression.

机译:通过D3.49(164)而非D3.32(147):D3.49(164)的突变,对阿片类阿片受体的组成性激活对于稳定受体的非活性形式及其表达至关重要。

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摘要

The roles of conserved aspartates in the third transmembrane domain of the rat mu opioid receptor (RMOR) were explored with mutations of D3.32(147) and D3.49(164). D3.49(164) in the highly conserved DRY motif was mutated to 13 amino acids. Except for the D3.49(164)E mutant, each mutant displayed little or no detectable [(3)H]diprenorphine binding, and pretreatment with naloxone greatly enhanced binding. D3.49(164)H, -Q, -Y, -M, and -E mutants were further studied. D3.32(147) was substituted with A or N. All seven mutants exhibited similar binding affinities for the antagonist [(3)H]diprenorphine as the wild-type. The D3.49(164)H, -Q, -Y, and -M mutants, but not the D3.49(164)E and D3.32(147) mutants, exhibited enhanced basal [(35)S]GTPgammaS binding which was comparable to the maximally activated level of the wild-type and was related to expression levels. Naloxone, naltrexone, and naloxone methiodide significantly inhibited the basal [(35)S]GTPgammaS binding of the D3.49(164) mutants, indicating inverse agonist activities. Treatment of the D3.49(164)Y mutant with pertussis toxin greatly reduced the basal [(35)S]GTPgammaS binding, demonstrating constitutive activation of Galpha(i)/Galpha(o). The D3.49(164)H, -Y, -M, and -Q mutants had higher affinities for DAMGO than the wild-type, which were not significantly lowered by GTPgammaS. Thus, mutation of D3.49(164) to H, Y, M, or Q in RMOR resulted in receptor assuming activated conformations. In contrast, the D3.49(164)E mutant displayed significantly lower basal [(35)S]GTPgammaS binding and reduced affinity for DAMGO. Upon incubation of membranes at 37 degrees C, the constitutively active D3.49(164)Y mutant was structurally less stable, whereas the inactivated D3.49(164)E mutant was more stable, than the wild-type. Computational simulations showed that the E3.49 side chain interacted strongly with the conserved R3.50 in the DRY motif and stabilized the inactive form of the receptor. Taken together, these results indicate that D3.49 plays an important role in constraining the receptor in inactive conformations.
机译:探讨了D3.32(147)和D3.49(164)突变在大鼠μ阿片受体(RMOR)的第三个跨膜域中的保守天冬氨酸的作用。高度保守的DRY基序中的D3.49(164)被突变为13个氨基酸。除D3.49(164)E突变体外,每个突变体均显示出很少或没有可检测到的[(3)H] diprenorphine结合,并且用纳洛酮预处理大大增强了结合。 D3.49(164)H,-Q,-Y,-M和-E突变体被进一步研究。 D3.32(147)被A或N取代。所有七个突变体都显示出与野生型拮抗剂[(3)H] diprenorphine相似的结合亲和力。 D3.49(164)H,-Q,-Y和-M突变体,而不是D3.49(164)E和D3.32(147)突变体,表现出增强的基础[(35)S] GTPgammaS结合它与野生型的最大活化水平相当,并且与表达水平有关。纳洛酮,纳曲酮和纳洛酮甲硫醚显着抑制D3.49(164)突变体的基础[(35)S] GTPgammaS结合,表明其反向激动剂活性。用百日咳毒素治疗D3.49(164)Y突变体大大降低了基础[(35)S] GTPgammaS的结合,证明了Galpha(i)/ Galpha(o)的组成型激活。 D3.49(164)H,-Y,-M和-Q突变体对DAMGO的亲和力高于野生型,而GTPgammaS并未显着降低其亲和力。因此,RMOR中D3.49(164)突变为H,Y,M或Q导致受体呈激活构象。相反,D3.49(164)E突变体显示出显着较低的基础[(35)S] GTPgammaS绑定和减少对DAMGO的亲和力。在37℃下孵育膜后,组成型活性D3.49(164)Y突变体在结构上较不稳定,而灭活的D3.49(164)E突变体则比野生型更稳定。计算模拟表明,E3.49侧链与DRY基序中的保守R3.50强烈相互作用,并稳定了受体的非活性形式。综上,这些结果表明,D3.49在限制受体处于非活性构象中起着重要作用。

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