In this study, differential gene expression between normal human mammary epithelial cells and their malignant counterparts (eight well established breast cancer cell lines) was studied using Incyte GeneAlbum 1–6, which contains 65,873 cDNA clones representing 33,515 individual genes. 3,152 cDNAs showed a ≥3.0-fold expression level change in at least one of the human breast cancer cell lines as compared with normal human mammary epithelial cells. Integration of breast tumor gene expression data with the genes in the tumor suppressor p53 signaling pathway yielded 128 genes whose expression is altered in breast tumor cell lines and in response to p53 expression. A hierarchical cluster analysis of the 128 genes revealed that a significant portion of genes demonstrate an opposing expression pattern, i.e. p53-activated genes are down-regulated in the breast tumor lines, whereas p53-repressed genes are up-regulated. Most of these genes are involved in cell cycle regulation and/or apoptosis, consistent with the tumor suppressor function of p53. Follow-up studies on one gene, RAI3, suggested that p53 interacts with the promoter of RAI3 and repressed its expression at the onset of apoptosis. The expression of RAI3 is elevated in most tumor cell lines expressing mutant p53, whereas RAI3 mRNA is relatively repressed in the tumor cell lines expressing wild-type p53. Furthermore, ectopic expression of RAI3 in 293 cells promotes anchorage-independent growth and small interfering RNA-mediated depletion of RAI3 in AsPc-1 pancreatic tumor cells induces cell morphological change. Taken together, these data suggest a role for RAI3 in tumor growth and demonstrate the predictive power of integrative genomics.

Orphan G-protein-coupled receptors are a large class of receptors whose cognate ligands are unknown. SP9155 (also referred to as AQ27 and GPR103) is an orphan G-protein-coupled receptor originally cloned from a human brain cDNA library. SP9155 was found to be predominantly expressed in brain, heart, kidney, retina, and testis. Phylogenetic analysis shows that SP9155 shares high homology with Orexin, NPFF, and cholecystokinin (CCK) receptors, but identification of the endogenous ligand for SP9155 has not been reported. In this study, we have used a novel method to predict peptides from genome data bases. From these predicted peptides, a novel RF-amide peptide, P52 was shown to selectively activate SP9155-transfected cells. We subsequently cloned the precursor gene of the P52 ligand and characterized the activity of other possible peptides encoded by the precursor. This revealed an extended peptide, P518, which exhibited high affinity for SP9155 (EC50 = 7 nm). mRNA expression analysis revealed that the peptide P518 precursor gene is predominantly expressed in various brain regions, coronary arteries, thyroid and parathyroid glands, large intestine, colon, bladder, testes, and prostate. These results indicate the existence of a novel RF-amide neuroendocrine peptide system, and suggest that SP9155 is likely the relevant G-protein-coupled receptor for this peptide.

We have identified an orphan G protein-coupled receptor, SP174, that shares a high degree of homology with the recently described ADP receptor P2Y(12). mRNA for SP174 is abundant in the brain and in cells of the immune system. In the present study, we demonstrate that SP174 is also a receptor for ADP, which is coupled to Galphai. ADP potently stimulates SP174 with an EC(50) of 60 nM, and other related nucleotides are active as well, with a rank order of potency 2-methylthio-ADP tetrasodium = adenosine 5'-O-2-(thio)diphosphate = 2-methylthio-ATP tetrasodium > ADP > AP3A >ATP > IDP. This pharmacological profile is similar to that for P2Y(12). We have also identified the murine homolog of SP174, which exhibits 75% homology to the human receptor. ADP is also a potent agonist at the murine receptor, and its pharmacological profile is similar to its human counterpart, but ADP and related nucleotides are more potent at the murine receptor than the human receptor. In keeping with the general nomenclature for the purinergic receptors, we propose designating this novel receptor P2Y(13).

P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to G alpha (i) have been described but have not yet been cloned, SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a G alpha (i)-coupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca2+ mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric G alpha subunits (G alpha (q/i)), A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC50 of 60 nM. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP = 2-MeS-ADP > ADP = adenosine 5'-O-2-(thio)diphosphate > 2-Cl-ATP > adenosine 5'-O-(thiotriphosphate). Thus, SP1999 is a novel, G alpha (i)-linked receptor for ADP.