Atwood et al. BMC Genomics 2011, 12:14 http://www.biomedcentral.com/1471-2164/12/14 receptors (figure 4A). Like HEK293 cells, AtT20 cells express mRNA for GABABR1 (with a detectable, but not significant level of GABABR2 mRNA), but unlike HEK293s they express several metabotropic glutamate receptor genes (figure 4B). For Class F/S in AtT20s we detected mRNA for six frizzled receptors but not smoothened receptor mRNA (figure 4C). AtT20’s have a somewhat sparser repertoire of GPCR signaling-related gene products than HEK293s, especially within the G protein subunits (figures 5 and 6). We next examined BV2 cells, a cell line that is often used as a model system for the study of microglial func- tion [32]. As shown in figures 1 and 2, BV2 cells predo- minately possess mRNA for anaphylatoxin, chemokine, and prostanoid Class A GPCRs as would be expected from an immune system-derived cell line. They also express mRNA for a scattering of other receptor types, such as a few muscarinic, somatostatin, and lipid recep- tors, among others. As has been reported in the litera- ture, BV2 cells express CB2 cannabinoid receptors (Cnr2) [33], which makes them unique among the other cell lines examined here. We also observed the expres- sion of a large number of different Class A orphan GPCR mRNAs as seen in figure 3, though a much less diverse repertoire of orphans from Class B (figure 4A) or Class C (figure 4B) receptors were present. BV2 cells express only one Class C GPCR at a significant, albeit, low mRNA level: the metabotropic glutamate receptor, mGluR1 (Grm1). Figure 4C shows that BV2 cells pos- sess mRNA for a few different types of frizzled receptors as well as the smoothened receptor. BV2 cells have a wide complement of GPCR signaling-related gene pro- ducts (figures 5 and 6). Though not quite as extensive as HEK293 cells, BV2 cells possess numerous isoforms of different phospholipases, PKA, PKC, and adenylyl cyclase. Their beta-arrestin, RGS protein, and GRK levels suggest the capacity to highly regulate GPCR sig- naling as well. Lastly we investigated the N18 cell line. This cell line is derived from a neuroblastoma and is commonly employed as a neuron-like cell line. It has been used as a tool to analyze ion channel function in response to toxins and other regulators [19,34-37]. We found that N18 cells have mRNA for a broad range of different Class A GPCRs as seen in figures 1 and 2. N18 cells have mRNA for a few types of muscarinic acetylcholine, adenosine, chemokine, neuropeptides, prostanoids, pro- tease-activated, and somatostatin receptors with a few other individual types such as CB1 cannabinoid recep- tors (Cnr1). Like the BV2 cells, N18 cells have mRNA for quite a few Class A orphan GPCRs (figure 3) with a similar profile as well. They further express mRNA for a broad complement of Class B GPCRs (figure 4A) but express a limited profile of Class C GPCRs (figure 4B). Page 8 of 14 They also have mRNA for a number of different types of frizzled receptors, but lack mRNA for a smoothened receptor (figure 4C). N18 cells express high levels of many of the GPCR signaling-related gene products. They have abundant levels of G proteins and numerous isoforms of PKA. N18 cells’ GPCRs are also presumably tightly regulated based on the assortment of GRK, RGS and beta-arrestin mRNAs detected (figures 5 and 6). In order to validate our findings with the microarray analyses, we performed quantitative real-time PCR (qPCR) analysis on these four cell lines. We chose a range of GPCRs, mostly orphan receptors (Cnr1, Cnr2, Gpr3, Gpr12, Gpr35, Gpr83, and Gpr119), as well as two phospholipases (Plcb1 and Plcb4) as candidates to measure their mRNA levels using this alternative method. We selected genes that had distinct and dif- ferential expression profiles across the four cell lines. The data are plotted in figure 7 in a manner that cor- relates the copy number of mRNAs measured in each cell line using qPCR with the levels of mRNA deter- mined from our microarray experiments. The qPCR data were broadly consistent with our microarray data. We observed higher copy numbers for all genes for which we had observed statistically significant microar- ray expression, suggesting an overall good correlation between our microarray and qPCR data. The qPCR did detect mRNA for a few genes whose levels of expres- sion did not reach statistical significance in the micro- array analyses. These genes are Plcb4, Gpr3, and Gpr92 in AtT20 cells and Plcb1, Gpr83 and Gpr92 in N18 cells. Discussion The ability to induce expression of a known protein in a model cellular system, such as the cell lines analyzed here, has served as a remarkable, vital tool. However, the GPCR research community that makes use of these cell lines has had only limited data on the complement of GPCRs and related signaling machinery expressed in them. When a cell line is used for heterologous expres- sion, ignorance of the endogenous protein expression profile of a cell line may lead to false conclusions about the nature of receptor function. Indeed, the cellular milieu can have a dramatic impact on GPCR signaling (see [22]. Microarray technology is now sufficiently accessible and mature to determine the mRNA expres- sion for cell lines from species whose genomes have been sequenced. Yet even where microarray studies have been conducted, their data remains only nominally accessible to those with sufficient means, experience and time to extract useful information. The absence of a systematic published study of mRNA expression for signaling proteins of general interest hinders research using these important cell lines. This study provides