Expression of human cysteinyl leukotriene receptor type 2: Using bacterial, yeast and baculovirus/insect cell expression systems.

摘要

The cysteinyl leukotrienes are members of the eicosanoid family. They are primary mediators in inflammatory diseases, such as asthma, and contribute to airway hyperresponsiveness, inflammation and obstruction. They have also have been implicated in the development of cardiovascular disease including atherosclerosis, myocardial infarction and stroke. Cysteinyl leukotrienes exert their effects via two known G protein-coupled receptors (GPCRs), cysteinyl leukotriene receptor type 1 and cysteinyl leukotriene receptor type 2 (known as CysLT1 and CysLT2, respectively). The CysLT 1 receptor has been characterized extensively but much less is known about the biological functions and signalling of CysLT2. Because GPCRs are naturally expressed in small amounts a plan to overexpress CysLT 2 was devised. The first expression system tested was codon-optimized human CysLT2 in bacteria in a manner analogous to the previously reported successful expression of the leukotriene receptor BLT1. Since this method did not produce satisfactory results, eukaryotic expression systems, in particular, the yeast and the baculovirus/insect cell systems were adopted. Both of these expression systems support post-translation modifications that should yield proper receptor folding. However, neither of these systems supported CysLT2 production in sufficient amounts. Further analysis of the baculovirus/insect cell system indicated that CysLT2 mRNA was produced but there were no signs of functional receptor as confirmed by a radioligand binding assay using a positive control (human embryonic kidney 293 cells transfected with a CysLT2 plasmid. New strategies for overexpression of human CysLT2 will be necessary to gain further insight into the biochemical properties of this receptor.