最近发现了两个DRD2突变,c.634A>T,p.Ile212Phe和c.1121T>G,p.Met374Arg,引起具有重叠特征但严重程度明显不同的多动运动障碍。Met374Arg变种的两个已知携带者有儿童早期疾病发作和更严重的运动,认知,和神经精神缺陷比任何已知的Ile212Phe变体携带者都多,其症状在青春期首先明显。这里,我们评估了培养细胞中两种变体功能的差异是否可以解释不同的致病性。两种变体的表达量均低于野生型受体,并表现出激动剂诱导的抑制蛋白结合的丧失,但变体之间的表达和抑制蛋白结合差异很小。基础和激动剂诱导的异源三聚体Gi/o/z蛋白的激活,然而,显示明显的差异;激动剂通常在Met374Arg比在Ile212Phe或野生型变体更有效。此外,与Ile212Phe相比,Met374Arg对所有测试的Gα亚型的组成激活更多。Met374Arg比Ile212Phe或野生型D2受体产生更大的环状AMP积累组成型抑制。Met374Arg和Ile212Phe比野生型D2受体对热灭活更敏感,正如其他组成型活性受体的报道,但Ile212Phe比Met374Arg受到的影响更大。额外的药理学表征表明,突变差异影响激动剂结合袋的形状和多巴胺的效力,去甲肾上腺素,还有酪胺.分子动力学模拟为增强的组成型激活和激动剂效力提供了结构原理。增强的组成型和激动剂诱导的G蛋白介导的信号传导可能有助于这些新变体的致病性。
Two recently discovered DRD2 mutations, c.634A > T, p.Ile212Phe and c.1121T > G, p.Met374Arg, cause hyperkinetic movement disorders that have overlapping features but apparently differ in severity. The two known carriers of the Met374Arg variant had early childhood disease onset and more severe motor, cognitive, and neuropsychiatric deficits than any known carriers of the Ile212Phe variant, whose symptoms were first apparent in adolescence. Here, we evaluated if differences in the function of the two variants in cultured cells could explain differing pathogenicity. Both variants were expressed less abundantly than the wild type receptor and exhibited loss of agonist-induced arrestin binding, but differences in expression and arrestin binding between the variants were minor. Basal and agonist-induced activation of heterotrimeric Gi/o/z proteins, however, showed clear differences; agonists were generally more potent at Met374Arg than at the Ile212Phe or wild type variants. Furthermore, all Gα subtypes tested were constitutively activated more by Met374Arg than by Ile212Phe. Met374Arg produced greater constitutive inhibition of cyclic AMP accumulation than Ile212Phe or the wild type D2 receptor. Met374Arg and Ile212Phe were more sensitive to thermal inactivation than the wild type D2 receptor, as reported for other constitutively active receptors, but Ile212Phe was affected more than Met374Arg. Additional pharmacological characterization suggested that the mutations differentially affect the shape of the agonist binding pocket and the potency of dopamine, norepinephrine, and tyramine. Molecular dynamics simulations provided a structural rationale for enhanced constitutive activation and agonist potency. Enhanced constitutive and agonist-induced G protein-mediated signaling likely contributes to the pathogenicity of these novel variants.