Department of Neuroscience and Pharmacology, The Panum Institute, Copenhagen University, Denmark.
Br J Pharmacol. 2010 Mar;159(5):1092-105. doi: 10.1111/j.1476-5381.2009.00633.x. Epub 2010 Feb 8.
In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthermore uncovered and characterized constitutive activity of both isoforms.
Expression levels of the hGPR17 isoforms were determined in several brain regions as well as heart and kidney using quantitative RT-PCR. A CREB reporter assay and [(35)S]-GTPgammaS binding were employed to assess the constitutive activity and the activation by UDP, UDP-glucose and -galactose and the cysteinyl leukotrienes LTC(4) and LTD(4). Leukotriene binding and induction of internalization were furthermore tested using homologous competition binding and antibody-feeding experiments respectively.
The short isoform (hGPR17-S) was expressed more abundantly (eight- to 23-fold) in the brain than the long isoform (hGPR17-L), whereas the opposite was observed in heart and kidney. As previously reported, the uracil nucleotides activated hGPR17-S with micromolar potencies. However, much lower potencies were observed for hGPR17-L with a 50- to 170-fold increase in EC(50). Furthermore, contrary to previous reports, neither of the isoforms was activated or bound by the cysteinyl leukotrienes. Finally, both receptors were demonstrated to be constitutively active through Galpha(i).
We present the first isoform-specific characterization of GPR17 and show that differences exist between the isoforms, in both expression pattern and pharmacological profile. In turn, our results indicate that the two human isoforms might serve tissue-specific functions.
在人类和非人类灵长类动物中，7TM 受体 GPR17 存在两种仅在 N 末端长度上有所不同的同工型。到目前为止，仅对短同工型进行了特征描述。因此，我们研究了两种剪接变体的基因表达和配体结合谱，并且还发现并描述了两种同工型的组成型活性。
使用定量 RT-PCR 测定几种脑区、心脏和肾脏中 hGPR17 同工型的表达水平。采用 CREB 报告基因测定和 [(35)S]-GTPγS 结合测定来评估 UDP、UDP-葡萄糖和 UDP-半乳糖以及半胱氨酰白三烯 LTC(4)和 LTD(4)对组成型活性和激活的作用。此外，还使用同源竞争结合和抗体喂养实验分别测试了白细胞三烯的结合和内化诱导。
短同工型（hGPR17-S）在脑中的表达丰度（8 至 23 倍）高于长同工型（hGPR17-L），而在心脏和肾脏中则相反。如前所述，尿嘧啶核苷酸以微摩尔的效力激活 hGPR17-S。然而，对于 hGPR17-L，EC50 增加了 50 至 170 倍，观察到的效力要低得多。此外，与之前的报道相反，两种同工型都没有被半胱氨酰白三烯激活或结合。最后，通过 Galpha(i)证明了两种受体均具有组成型活性。
我们首次对 GPR17 进行了同工型特异性特征描述，并显示两种同工型在表达模式和药理学特征上存在差异。反过来，我们的结果表明，两种人类同工型可能具有组织特异性功能。
