Nielsen S M, Elling C E, Schwartz T W
Department of Pharmacology, Panum Institute, University of Copenhagen, Denmark.
Eur J Biochem. 1998 Jan 15;251(1-2):217-26. doi: 10.1046/j.1432-1327.1998.2510217.x.
Several membrane proteins have been functionally expressed from non-covalently coupled, contiguous segments especially with the split-site located between natural domains. Experiments using such 'split-proteins' were here performed in the tachykinin neurokinin-1 (NK1) receptor with co-expression of contiguous segments with split-sites positioned in various intracellular and extracellular loops. The construct where the split-site was located in intracellular loop 3 gave a reasonable expression level of substance-P-binding sites, i.e. 12% of wild-type expression. Of the other split-receptors tested, only the one with the split-site located just outside transmembrane (TM) segment-V gave any detectable substance P binding, which however only was 1% of the wild-type expression level. The construct with the split-site located in intracellular loop 3 bound all of the tested peptide agonists and non-peptide antagonists with normal affinity and was able to stimulate inositol phosphate turnover with a normal EC50 for substance P and an Emax according to the expression level. When intracellular loop 3 was either extended with 112 amino acid residues derived from the muscarine M2 receptor or, when major parts of the loop were deleted in the non-split NK1 receptor, the affinity for neither substance P nor for the prototype nonpeptide antagonist, CP96,345 was affected, yet an increase in EC50 for substance P was observed. Also in the split-receptor, most of intracellular loop 3 could be substituted or even deleted without affecting ligand affinity, although a decreased expression level was observed in constructs having major deletions. It is concluded, that the NK1 receptor is preferentially reconstituted by co-expression of a putative A-domain including TM-I-V and a B-domain including TM-VI and -VII. It is suggested that a number of rhodopsin-like 7TM receptors may function as two-domain structures based on the finding that a network of short loops has been highly conserved within each of the putative domains and, that these domains are separated by a relatively long and in respect of length poorly conserved loop, i.e. intracellular loop 3.
几种膜蛋白已从非共价偶联的连续片段中实现功能表达，特别是分裂位点位于天然结构域之间。在此，利用速激肽神经激肽-1（NK1）受体进行了使用此类“分裂蛋白”的实验，共表达了分裂位点位于不同细胞内和细胞外环的连续片段。分裂位点位于细胞内环3的构建体产生了合理水平的P物质结合位点表达，即野生型表达的12%。在测试的其他分裂受体中，只有分裂位点位于跨膜（TM）片段-V外侧的那个受体有任何可检测到的P物质结合，但这仅为野生型表达水平的1%。分裂位点位于细胞内环3的构建体以正常亲和力结合所有测试的肽激动剂和非肽拮抗剂，并且能够以正常的P物质EC50和根据表达水平的Emax刺激肌醇磷酸周转。当细胞内环3用源自毒蕈碱M2受体的112个氨基酸残基进行延伸时，或者当在非分裂的NK1受体中该环的大部分被缺失时，对P物质和原型非肽拮抗剂CP96,345的亲和力均未受影响，但观察到P物质的EC50增加。同样在分裂受体中，细胞内环3的大部分可以被替代甚至缺失而不影响配体亲和力，尽管在有大量缺失的构建体中观察到表达水平降低。得出的结论是，NK1受体优先通过共表达包括TM-I-V的假定A结构域和包括TM-VI和-VII的B结构域来重构。基于以下发现提出，许多视紫红质样7TM受体可能作为双结构域发挥作用：在每个假定结构域内，短环网络高度保守，并且这些结构域由一个相对较长且长度保守性较差的环（即细胞内环3）分隔。
