Gα protein selectivity determinant specified by a viral chemokine receptor-conserved region in the C tail of the human herpesvirus 8 G protein-coupled receptor
Gα protein selectivity determinant specified by a viral chemokine receptor-conserved region in the C tail of the human herpesvirus 8 G protein-coupled receptor
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DOI：
10.1128/jvi.78.5.2460-2471.2004
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发表时间：
2004-03-01
影响因子：
5.4
通讯作者：
Nicholas, J
中科院分区：
文献类型：
--
作者：
Liu, CQ;Sandford, G;Nicholas, J
由人类疱疹病毒8型（HHV-8）开放阅读框74（ORF 74）特异性的病毒G蛋白偶联受体（vGPCR）是配体非依赖性趋化因子受体，其在其他特征性γ疱疹病毒和β疱疹病毒中的相关受体中具有结构和功能同源物。γ疱疹病毒vGPCR的序列比较显示，在C尾的高度保守的区域，只是远端的第七个跨膜结构域。对HHV-8 ORF 74的相应密码子进行突变，以提供C-尾改变的蛋白用于功能分析。通过测量受体活化的血管内皮生长因子启动子诱导和NF-κ B，促分裂原活化蛋白激酶，和Ca 2+信号，我们发现，虽然一些改变的受体表现出一般的信号转导缺陷，其他人有不同的激活配置文件，提示选择性Get蛋白偶联。这得到以下发现的支持：vGPCR和代表性的功能改变的变体vGPCR. 8（R322 W）和vGPCR. 15（M325 S）受到Ga（i）（百日咳毒素）、蛋白激酶C（GF 109203 X）和磷脂酰肌醇3-激酶（渥曼青霉素）抑制剂的不同影响。与信号传导数据一致，[S-35] GTP γ S掺入试验显示vGPCR. 15与Ga（q）优先偶联，VGPCR. 8不能与Ga（q）功能性偶联。然而，这两种变体，野生型vGPCR和受体的C尾缺失形式同样能够与Ga（q）物理缔合。结合，我们的数据表明，HHV-8 vGPCR包含离散的网站的G α相互作用和受体残基在近端区域的胞质尾是G α蛋白偶联特异性的决定因素。
The viral G-protein coupled receptor (vGPCR) specified by human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is a ligand-independent chemokine receptor that has structural and functional homologues among other characterized gammaherpesviruses and related receptors in the betaherpesviruses. Sequence comparisons of the gammaherpesvirus vGPCRs revealed a highly conserved region in the C tail, just distal to the seventh transmembrane domain. Mutagenesis of the corresponding codons of HHV-8 ORF74 was carried out to provide C-tail-altered proteins for functional analyses. By measuring receptor-activated vascular endothelial growth factor promoter induction and NF-kappaB, mitogen-activated protein kinase, and Ca2+ signaling, we found that while some altered receptors showed general signaling deficiencies, others had distinguishable activation profiles, suggestive of selective Get protein coupling. This was supported by the finding that vGPCR and representative functionally altered variants, vGPCR.8 (R322W) and vGPCR.15 (M325S), were affected differently by inhibitors of Galpha(i) (pertussis toxin), protein kinase C (GF109203X), and phosphatidylinositol 3-kinase (wortmannin). Consistent with the signaling data, [S-35]GTPgammaS incorporation assays revealed preferential coupling of vGPCR.15 to Galpha(q) and an inability of VGPCR.8 to couple functionally to Galpha(q). However, both variants, wild-type vGPCR, and a C-tail deletion version of the receptor were equally able to associate physically with Galpha(q). Combined, our data demonstrate that HHV-8 vGPCR contains discrete sites of Galpha interaction and that receptor residues in the proximal region of the cytoplasmic tail are determinants of Galpha protein coupling specificity.