Atwood et al. BMC Genomics 2011, 12:14 http://www.biomedcentral.com/1471-2164/12/14 Each subunit couples to specific cellular protein targets (e.g. adenylyl cyclases) through which cellular activity is modulated. The Gb/g G protein subunits are capable of acting at their own specific targets, such as modulating ion channel activity [9-11]. The available complement of heterotrimeric G proteins will therefore affect its response profile. A regulatory system controls the timing and duration of GPCR signaling. GPCR kinases (GRKs) phosphorylate agonist-occupied receptors, typically leading to receptor desensitization. Beta-arrestins are scaffolding proteins that are recruited to activated GPCRs and prevent G protein association with the receptor. They also serve to spatially segregate GPCR signaling to molecules such as mitogen-activated protein kinases [12,13]. The Regula- tors of G protein signaling (RGS) proteins, which enhance the GTPase activity of Ga subunits, is another large family of proteins important in fine-tuning GPCR signaling [14,15]. Due to their central roles in many cellular functions, GPCRs are important therapeutic targets. Different reports estimate that between 30 and 50% of all pre- scription drugs target GPCRs. Interestingly, these drugs only target ~10% of the non-chemosensory receptors, leaving hundreds of GPCRs as potential drug targets [2,9,16]. Identifying ligands, studying receptor protein interac- tions, and measuring cellular effects of receptor activation is often achieved by heterologous expression of GPCRs in cell lines such as HEK293 and AtT20 [17,18]. They also serve as models of cell types from which they are drawn (e.g. BV2 for microglia and N18 for neurons) [19-21]. However these cell lines have remained something of a black box; researchers make use of them without full knowledge of the complement of GPCRs and related pro- teins. The presence of an unknown protein in these cell lines may confound an otherwise sound series of experi- ments and differential expression of GPCRs and their regulatory proteins may explain differences in reported results across cell lines [13,17,22-25]. It is also clear that GPCRs can form oligomers with other GPCRs. These oli- gomers may confer different signaling properties to a transfected GPCR of interest, and can add layers of varia- bility and complexity. Efforts to define expression of GPCRs, on a receptor- by-receptor basis, using polymerase chain reaction (PCR)-based methods have only scratched the surface. Even for the widely used HEK293 cells, the expression of fewer than thirty GPCRs has been described [17,26]. Recently developed high-throughput methods have made possible the simultaneous assay of mRNA levels for thousands of genes. Microarray analyses have matured sufficiently in terms of accessibility and cost to make their application to this question tractable. We are Page 2 of 14 not the first to make use of microarray technology to examine mRNA expression levels in HEK293 cells [26], however, most studies that have used microarray ana- lyses on these cell lines have examined specific gene products of interest and reported data only for those gene products [20,26-28]. Their full data sets are avail- able through NCBI, but are difficult for the non-specia- list user to access and collate. As a consequence, we sought to identify and make accessible the full complement of endogenously expressed non-chemosensory GPCRs of four cell lines used in GPCR research: the HEK293, AtT20, BV2 and N18 lines. Using microarray analysis we measured the mRNA levels from each of these four cell lines specifi- cally identifying the expression levels of GPCRs and over a hundred of GPCR-related gene products and report them here. Results We first examined HEK293 cells, a cell line that was made by transforming human embryonic kidney cells with adenovirus type 5 DNA. Developed in 1977, HEK293 cells may be the single most widely used cell line for heterologous expression (both transient and stable expression) of GPCRs and other proteins of inter- est [17]. Figures 1,2,3,4,5 and 6 show the mRNA levels of GPCR and GPCR-related gene products found in HEK293 cells. Of Class A GPCRs, shown in figures 1 and 2, HEK293 cells express mRNA for several different types of receptors within specific families of receptors: multiple types of adenosine, lysophospholipid, puriner- gic, prostanoid, and protease-activated receptors, among others. In results relevant to the study of orphan GPCRs (figure 3), HEK293 cells contain high message levels of GPR37, 2 types of orphan-LGR (glycoprotein hormone) receptors–LGR4 and LGR5, GPR39–and very high levels of the melatonin-related receptor GPR50. HEK293 cells may therefore be suitable tools for deorphanization of these receptors. Interestingly, HEK293 cells contain rela- tively little mRNA for Class B GPCRs with known ligands (figure 4A), but quite high levels of many orphan Class B GPCRs. The same is true for Class C GPCRs as seen in figure 4B though they do appear to contain high levels of GABABR1 receptor message, though lacking mRNA for GABABR2, necessary to form a fully func- tional GABA-B receptor. Finally, for GPCRs, HEK293 cells contain a number of types of frizzled receptors as well as smoothened receptor (figure 4C). Figures 5 and 6 show the mRNA levels of various types of GPCR sig- naling-related gene products such as the G proteins and a number of their targets and downstream effectors as well as proteins that regulate GPCR signaling, the GRK, beta-arrestin (ARRB) and RGS proteins. Interestingly HEK293 cells express mRNA for numerous isoforms of