Johard H A, Muren J E, Nichols R, Larhammar D S, Nässel D R
Department of Zoology, Stockholm University, SE-10691, Stockholm, Sweden.
Brain Res. 2001 Nov 16;919(1):94-105. doi: 10.1016/s0006-8993(01)03004-9.
Tachykinins constitute a neuropeptide family that mediate their actions via a subfamily of structurally related G-protein-coupled receptors. Two receptors, Drosophila neurokinin receptor (NKD) and Drosophila tachykinin receptor (DTKR), with sequence similarities to mammalian tachykinin receptors have previously been cloned in Drosophila. In this study we have isolated a cockroach (Leucophaea maderae) cDNA clone by screening a brain cDNA library with a degenerate oligonucleotide probe based on a conserved sequence within the seventh transmembrane region of the Drosophila tachykinin receptors. This clone, Leucophaea tachykinin receptor (LTKR), encodes a portion of a putative receptor which could be aligned with the C-terminal half of members of the tachykinin receptor subfamily. In the fifth, sixth and seventh transmembrane regions the deduced amino acid sequence of LTKR exhibits 79% sequence identity to the DTKR receptor and 54% to that of NKD. This suggests that LTKR is orthologous to the DTKR receptor. To study the distribution of the predicted LTKR protein by immunocytochemistry, antisera were raised against synthetic peptides corresponding to a region of the third intracellular loop of LTKR. In the cockroach brain immunoreactive neuronal processes were seen in several synaptic neuropils of the protocerebrum and tritocerebrum as well as in the frontal ganglion. Some immunoreactive neuronal cell bodies were detected in the protocerebrum. Double labeling immunocytochemistry revealed that there is a substantial superposition between distribution of LTKR and processes containing tachykinin-related peptide (TRP). Some brain areas, however, only display TRP immunoreactive processes and no LTKR, suggesting the presence of at least one more TRP receptor type.
速激肽构成一个神经肽家族，它们通过结构相关的G蛋白偶联受体亚家族介导其作用。此前已在果蝇中克隆出两种与哺乳动物速激肽受体具有序列相似性的受体，即果蝇神经激肽受体（NKD）和果蝇速激肽受体（DTKR）。在本研究中，我们基于果蝇速激肽受体第七跨膜区域内的保守序列，用简并寡核苷酸探针筛选脑cDNA文库，分离出了一个蟑螂（ maderae）cDNA克隆。这个克隆，即蟑螂速激肽受体（LTKR），编码了一个假定受体的一部分，该部分可与速激肽受体亚家族成员的C端后半部分比对。在第五、第六和第七跨膜区域，LTKR推导的氨基酸序列与DTKR受体的序列一致性为79%，与NKD的序列一致性为54%。这表明LTKR与DTKR受体是直系同源的。为了通过免疫细胞化学研究预测的LTKR蛋白的分布，制备了针对与LTKR第三细胞内环区域相对应的合成肽的抗血清。在蟑螂脑中，在原脑和后脑的几个突触神经纤维网以及额神经节中都观察到了免疫反应性神经突起。在原脑中检测到了一些免疫反应性神经细胞体。双重标记免疫细胞化学显示，LTKR的分布与含有速激肽相关肽（TRP）的突起之间存在大量重叠。然而，一些脑区仅显示TRP免疫反应性突起，而没有LTKR，这表明至少还存在一种TRP受体类型。
