Identification of a tetramerization domain in the C terminus of the vanilloid receptor
Identification of a tetramerization domain in the C terminus of the vanilloid receptor
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DOI：
10.1523/jneurosci.0202-04.2004
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发表时间：
2004-06-09
影响因子：
5.3
通讯作者：
Ferrer-Montiel, A
中科院分区：
文献类型：
--
作者：
García-Sanz, N;Fernández-Carvajal, A;Ferrer-Montiel, A
TRPV1（瞬时受体电位香草酸受体亚型1）是由香草酸、质子和热量门控的TRP通道家族的成员。在结构上，TRPV1似乎是一个四聚体，由四个相同的亚基围绕中心水孔组装而成。控制其亚基寡聚化的分子决定因素仍然难以捉摸。在这里，我们报道了在TRPV1的C端鉴定了一个包含（684）Glu-(721)Arg的片段（称为trp样结构域）作为该蛋白的结合结构域（AD）。纯化后的重组TRPV1 C末端（TRPV1-C）在体外形成离散稳定的多聚体。酵母双杂交和下拉实验表明，当片段（684）Glu-(721)Arg被删除时，TRPV1-C的自结合被阻断。生化和免疫学分析表明，从全长TRPV1单体中去除AD可阻断与野生型TRPV1亚基稳定异聚体的形成。缺失无孔TRPV1亚基中的AD抑制了其强大的显性阴性表型。总之，这些发现与TRPV1中的trp样结构域是受体亚基四聚成功能通道的分子决定因素这一原则是一致的。我们的观察结果表明，TRP蛋白家族中的同源TRP结构域可能作为一个普遍的、进化保守的AD参与亚基多聚。
TRPV1(transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising (684)Glu-(721)Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment (684)Glu-(721)Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.