Analysis of endogenous S1P and LPA receptor expression in CHO-K1 cells
Introduction
Heterologous expression of G protein-coupled receptors (GPCRs) in mammalian cells is commonly used when studying the pharmacology and signal responsiveness of such proteins. Studies of this nature are performed using a wide variety of hosts, although cell lines such as the Chinese hamster ovary cell line CHO-K1, are extensively used due to their amenity to transfection and robustness in culture. This has made CHO-K1s a cell line of choice for GPCR ligand and drug screening activities.
A family of GPCRs, colloquially known as the Edg receptors, is the subject of much current investigation (reviewed by Fukushima and Chun, 2001, Kluk and Hla, 2002, Takuwa et al., 2002). The Edg receptor family was recently reclassified into two clusters based on ligand selectivity: S1P1/2/3/4/5 (formerly Edg1/5/3/6/8) specifically respond to S1P whilst LPA1/2/3 (formerly Edg2/4/7) respond selectively to LPA (Chun et al., 2002). Expression of S1P and LPA receptor genes is widespread and their presence in many of the host cell lines which are commonly used for expression of recombinant receptors can lead to interfering signals in in-vitro assays. Cell lines which are unresponsive to S1P and LPA in a variety of assays, have been identified (e.g. RH7777; Zhang et al., 1999), however studies of the S1P4 receptor undertaken in our laboratories failed to detect activation of this receptor expressed in RH7777 cells (unpublished results). In contrast, when S1P4 was expressed in CHO-K1 cells the receptor was constitutively active and could be further stimulated by S1P, as detected using [35S]GTPγS binding assay (G. Holdsworth et al., manuscript in preparation). However, untransfected CHO-K1 cells are known to respond to S1P and LPA in certain in-vitro assays (e.g. calcium flux (Yamazaki et al., 2000; our unpublished observations)), presumably as a consequence of endogenous S1P and LPA receptor expression. Although there is now at least one online database which details endogenous GPCR expression in commonly used cell lines such as CHO-K1s (http://www.tumor-gene.org/GPCR/gpcr.html) the information is often incomplete and of limited use to the researcher.
We identify herein which of the Edg family receptors were expressed in the CHO-K1 cell line. Similar investigations have been undertaken for other commonly used cell lines (Graeler et al., 1998, Motohashi et al., 2000, Fueller et al., 2003). Previous studies which examined expression of some of the Edg family receptors in this CHO-K1 cells by Northern blotting used rat or human probes (Okamoto et al., 1998, Kon et al., 1999, Sato et al., 1999a). Hence no definitive study of the S1P and LPA receptors expressed in this cell line had been undertaken. We used RT-PCR to profile the repertoire of S1P and LPA receptor transcripts expressed in the CHO-K1 cell line. Since no sequence information was available for Chinese hamster S1P and LPA receptors, semi-redundant oligonucleotide pairs were designed from alignments of human, rat and mouse S1P and LPA receptor sequences. A portion of the coding sequence of each of the eight Edg family receptors was then amplified from CHO-K1 genomic DNA and sequenced, thus providing novel sequence information for these Chinese hamster genes and allowing the design of species-specific oligonucleotide pairs. Finally, the species-specific oligonucleotides were used to show the presence of S1P1,2,4 and LPA1 transcripts in CHO-K1 mRNA.
Section snippets
Materials and methods
General laboratory chemicals were purchased from Sigma Aldrich, (Gillingham, Dorset, UK) or BDH, (Poole, Dorset, UK) unless stated otherwise. CHO-K1 cells were originally sourced from ATCC. Restriction endonucleases, dNTPs, DNA molecular weight markers and amplification grade deoxyribonuclease I were purchased from New England Biolabs, (Hitchin, Herts., UK) or Roche Diagnostics, (Lewes, East Sussex, UK). Advantage-GC 2 and Advantage RT-for-PCR kits were from Clontech Laboratories, (Palo Alto,
Amplification of gene fragments from Chinese hamster genomic DNA
The NCBI Entrez database was queried for sequences of the eight S1P and LPA receptors from human, rat and mouse. Alignments of protein and nucleotide coding sequences were generated for each receptor and areas of high inter-species similarity chosen as regions in which oligonucleotides could be designed. For each receptor, sequences were strongly conserved between species, although there were frequent silent changes at the 3-position within the nucleotide sequences across these species. Hence,
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