Michealraj K A, Jatana N, Narayanan L, Chattopadhyay A, Thelma B K
Department of Genetics, University of Delhi South Campus, New Delhi, India.
Bioinformatics Infrastructure Facility, Sri Venkateswara College, New Delhi, India.
Neuroscience. 2014 Mar 14;262:176-89. doi: 10.1016/j.neuroscience.2013.12.059. Epub 2014 Jan 6.
Next generation sequencing technologies have facilitated a notable shift from common disease common variant hypothesis to common disease rare variant, as also witnessed in recent literature on schizophrenia. Dopamine receptor D4 (DRD4), a G-protein-coupled receptor is associated with psychiatric disorders and has high affinity for atypical antipsychotic clozapine. We investigated the functional role of rare genetic variants in DRD4 which may have implications for translational medicine. CHO-K1 cells independently expressing four rare non-synonymous variants of DRD4 namely R237L, A281P, S284G located in the third cytosolic loop and V194G, located in the fifth transmembrane domain were generated. Their genotype-phenotype correlations were evaluated using [³H]spiperone binding, G-protein activation and molecular dynamics-simulation studies. A281P and S284G were functionally similar to wildtype (WT). With R237L, potency of dopamine and quinpirole reduced ∼sixfold and threefold respectively compared to WT; [³H]spiperone binding studies showed a reduction in total number of binding sites (∼40%) but not binding affinity, in silico docking studies revealed that binding of both dopamine and spiperone to R237L was structurally similar to WT. Of note, V194G variant failed to inhibit forskolin-stimulated adenylate cyclase activity and phosphorylate extracellular signal-regulated kinase; showed significant reduction in binding affinity (K(d)=2.16 nM) and total number of binding sites (∼66%) compared to WT in [³H]spiperone binding studies; and ligand docking studies showed that binding of dopamine and spiperone is superficial due to probable structural alteration. Transmembrane variant V194G in DRD4.4 results in functional alteration warranting continuing functional analysis of rare variants.
下一代测序技术推动了从常见疾病常见变异假说到常见疾病罕见变异的显著转变，这在近期关于精神分裂症的文献中也有体现。多巴胺受体D4（DRD4）是一种G蛋白偶联受体，与精神疾病相关，对非典型抗精神病药物氯氮平具有高亲和力。我们研究了DRD4中罕见基因变异的功能作用，这可能对转化医学有影响。生成了独立表达DRD4四个罕见非同义变异的CHO-K1细胞，这些变异分别是位于第三个胞质环的R237L、A281P、S284G以及位于第五个跨膜结构域的V194G。使用[³H]司哌罗 binding、G蛋白激活和分子动力学模拟研究评估了它们的基因型-表型相关性。A281P和S284G在功能上与野生型（WT）相似。对于R237L，与WT相比，多巴胺和喹吡罗的效力分别降低了约六倍和三倍；[³H]司哌罗 binding研究表明结合位点总数减少了约40%，但结合亲和力未降低，计算机对接研究显示多巴胺和司哌罗与R237L的结合在结构上与WT相似。值得注意的是，V194G变异未能抑制福司可林刺激的腺苷酸环化酶活性和磷酸化细胞外信号调节激酶；在[³H]司哌罗 binding研究中，与WT相比，结合亲和力（K(d)=2.16 nM）和结合位点总数显著降低（约66%）；配体对接研究表明，由于可能的结构改变，多巴胺和司哌罗的结合是表面的。DRD4.4中的跨膜变异V194G导致功能改变，需要对罕见变异进行持续的功能分析。
