Pharmacological analysis of CCK2 receptors up-regulated using engineered transcription factors
Introduction
The cholecystokinin 2 (CCK2, formerly CCKB) receptor is a guanine nucleotide coupled receptor (GPCR) that mediates the effects of the peptide hormones gastrin and cholecystokinin. The physiological responses to CCK2 receptor activation include gastric acid secretion, growth of gastrointestinal mucosa, along with effects on satiety and anxiety [1], [2], [3]. In addition, the CCK2 receptor has been implicated as a growth factor in some pathophysiological conditions including colorectal carcinoma [4] and Barrett's esophagus [5]. These findings indicate that the CCK2 receptor is a potential valuable therapeutic target and consequently a number of receptor selective ligands have been developed [6]. Prior to the cloning of the CCK2 receptor, the affinity of these ligands was routinely measured using animal tissue preparations (e.g. radioligand binding to the rat cerebral cortex membranes [7]). However, the finding that a single amino-acid substitution accounted for the reverse selectivity of the non-peptide antagonists L-365,260 and L-364,718 between dog and rat [8] highlighted the need for evaluating the affinity of potentially therapeutic compounds at the human receptor in early drug discovery programs.
To investigate ligand affinity at human receptors it is possible to use specimens of human tissue obtained from individual patients at surgery [9]. However, it is difficult to obtain ‘normal’ human tissue from surgical intervention and the success rate in conducting bioassay using human tissue preparations is notoriously low (see Refs. [9], [10], [11]). Alternatively, immortalized human cell lines that endogenously express the target gene can be used. This source of material is not optimal as these cell lines have generally been propagated from human carcinomas and, therefore, may contain mutated or altered signaling pathways that are associated with cancer cell growth. Therefore, the most common experimental method for the pharmacological characterization of GPCRs involves stable or transient transfection of cDNA into rapidly growing cells (for example COS-7 and CHO cells) to create a protein-expression system. Recently an alternative expression method has been developed where one can investigate proteins in their native environment by altering gene expression using engineered transcription factors [12]. The Cys2–His2 zinc finger proteins (ZFPs) have been shown to bind with high affinity to target DNA sequences and it has been demonstrated that coupling of these DNA binding proteins to characterized activator or repressor domains can up-regulate [13] or down-regulate [14] specific genes. The aim of this study was to pharmacologically characterize a human cell line which was engineered to express CCK2 receptors (HEKZFP cell line) and to see if there were any significant differences in the affinity of known CCK-receptor selective ligands.
In the HEKZFP cell line the CCK2 receptor expression was found to be increased ∼92-fold over background after de-repression of the zinc finger protein. The receptor protein was shown to be functional using calcium mobilization techniques and the agonist potency values obtained were consistent with the activation of the CCK2 receptor. In addition, seven, structurally diverse, CCK receptor selective antagonists were investigated with affinity values for the CCK2 receptor spanning five orders of magnitude. Overall, the affinity of these compounds at the CCK2 receptors expressed in HEKZFP cells was consistent with their affinity at cloned human CCK2 receptors. Two compounds, JB83182 and SR27897, did express approximately 0.8–1 log unit lower affinity in the HEKZFP compared to previous literature values, however, further research using human tissue is required to determine which expression system is more predictive of the human CCK2 receptors expressed physiologically.
Section snippets
Zinc finger protein expression system
Zinc Finger Proteins (ZFP) that are specific for the CCK2R gene were generated by Sangamo Biosciences and used to create a CCK2 receptor expression system in HEK cells (for details see Ref. [12]). In brief, CCK2 receptor specific zinc finger proteins were placed under the control of a tetracycline-regulatable promoter and transfected into the T-REx-293 cell line (which stably expresses the Tet-repressor; Invitrogen, Carlsbad, CA, USA). This cell expression system was termed the HEKZFP system.
Confirmation of the sequence identity of the CCK2 receptor expressed in the HEKZFP system and quantitation of the levels of CCK2 receptor expressed
The full length CCK2 receptor was amplified from total RNA extracted from HEKZFP cells treated with doxycycline. The PCR product was sequenced and was shown to have 100% homology to the human CCK2 receptor identified by Lee and co-workers [17]. Using real-time RT-PCR, the expression level of the CCK2 receptor was found to be 92 ± 15 fold greater in the HEKZFP cells that had been induced with 2 ng ml-1 doxycycline, compared to non-induced control HEKZFP cells. In addition, no amplified transcripts
Discussion
Using engineered zinc finger proteins that are selective for the CCK2 receptor, a cell system was created that expressed elevated levels of CCK2 receptors (for details see Ref. [12]). This system utilized a human cell line (HEK) as the host cell and, as such, provided a bioassay system where the CCK2 receptor was expressed in its native cellular context. In this study we have demonstrated that the CCK2 receptor up-regulated in the HEKZFP system has 100% nucleotide homology to the previously
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