Therapeutic angiogenesis is likely to require the administration of factors that complement each other. Activation of the receptor tyrosine kinase (RTK) Flk1 by vascular endothelial growth factor (VEGF) is crucial, but molecular interactions of other factors with VEGF and Flk1 have been studied to a limited extent. Here we report that placental growth factor (PGF, also known as PlGF) regulates inter- and intramolecular cross talk between the VEGF RTKs Flt1 and Flk1. Activation of Flt1 by PGF resulted in intermolecular transphosphorylation of Flk1, thereby amplifying VEGF-driven angiogenesis through Flk1. Even though VEGF and PGF both bind Flt1, PGF uniquely stimulated the phosphorylation of specific Flt1 tyrosine residues and the expression of distinct downstream target genes. Furthermore, the VEGF/PGF heterodimer activated intramolecular VEGF receptor cross talk through formation of Flk1/Flt1 heterodimers. The inter- and intramolecular VEGF receptor cross talk is likely to have therapeutic implications, as treatment with VEGF/PGF heterodimer or a combination of VEGF plus PGF increased ischemic myocardial angiogenesis in a mouse model that was refractory to VEGF alone.

Clones encoding a new human P2Y receptor, provisionally called P2Y 11 , have been isolated from human placenta complementary DNA and genomic DNA libraries. The 1113-base pair open reading frame is interrupted by one intron. The P2Y 11 receptor is characterized by considerably larger second and third extracellular loops than the subtypes described so far. The deduced amino acid sequence exhibits 33% amino acid identity with the P2Y 1 receptor, its closest homolog. Northern blot analysis detected human P2Y 11 receptor messenger RNA in spleen and HL-60 cells. The level of P2Y 11 transcripts was strongly increased in these cells after granulocyte differentiation induced by retinoic acid or dimethyl sulfoxide. The new receptor was stably expressed in 1321N1 astrocytoma and CHO-K1 cells, where it couples to the stimulation of both the phosphoinositide and adenylyl cyclase pathways, a unique feature among the P2Y family. The rank order of agonists potency was: ATP > 2-methylthio-ATP >>> ADP, whereas UTP and UDP were inactive, indicating that it behaves as a selective purinoceptor.An impressive number of P 2 receptor subtypes has been cloned since 1993. A molecular nomenclature has been established in which G protein-coupled P 2 receptors are named P2Y, whereas P 2 receptors having an intrinsic ion channel activity are named P2X (1, 2). The P2Y family encompasses selective purinoceptors (the P2Y 1 receptor (3, 4) activated preferentially by ADP and ATP), nucleotide receptors responsive to both adenine and uracil nucleotides (the P2Y 2 receptor (5, 6) activated equipotently by ATP and UTP, and the P2Y 8 receptor (7) activated equally by all triphosphate nucleotides), and pyrimidinoceptors (the P2Y 3 (8) and P2Y 6 (9 -11) receptors activated preferentially by UDP, and the P2Y 4 receptor (11-13) activated preferentially by UTP). All these P2Y subtypes are coupled exclusively to the phosphoinositide pathway. The inclusion of other receptors (P2Y 5 (14) and P2Y 7 (15)) within the P2Y family remains controversial (16 -18). Here we report the cloning of a new human gene encoding a G protein-coupled receptor that behaves as a selective purinoceptor and is coupled to the stimulation of both adenylyl cyclase and phospholipase C. 6 A, all-trans retinoic acid, and 12-Otetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma. 2-Methylthio-ATP (2MeSATP), 2-methylthio-ADP (2MeSADP) and 8-(p-sulfophenyl) theophylline were from Research Biochemicals International (Natick, MA). Forskolin was purchased from Calbiochem. (Bierges, Belgium). Indomethacin and Me 2 SO were from Merck. Rolipram was a gift from the Laboratoires Jacques Logeais (Trappes, France). The HL-60 human cell line was obtained from the American Type Culture Collection (Rockville, MD). The human genomic DNA library was from Stratagene (La Jolla, CA). The human placenta cDNA library was kindly given by Prof. P. Chambon (Strasbourg, France). pEFIN3 is an expression vector developed by Euroscreen (Brussels, Belgium). Multiple human tissue Northern blot (MTN) were from ...

We have cloned and expressed a novel human Gprotein-coupled receptor closely related to the human P2Y 12 receptor. It corresponds to the orphan receptor called GPR86. GPR86 proved to be a G i -coupled receptor displaying a high affinity for ADP, similar to the P2Y 12 receptor and can therefore be tentatively called P2Y 13 . In 1321N1 cells, the P2Y 13 receptor coupled to the phosphoinositide pathway only when coexpressed with G␣ 16 . Inositol trisphosphate formation was stimulated equipotently by nanomolar concentrations of ADP and 2MeSADP, whereas 2MeSATP and ATP were inactive. In CHO-K1 cells expressing the P2Y 13 receptor, ADP and 2MeSADP had a biphasic effect on the forskolin-stimulated accumulation of cAMP: inhibition at nanomolar concentrations and potentiation at micromolar levels. In the same cells, ADP and 2MeSADP also stimulated the phosphorylation of Erk1 and Erk2, in a pertussis toxinsensitive way. The tissue distribution of P2Y 13 was investigated by reverse transcriptase-polymerase chain reaction, and the predominant signals were obtained in spleen and brain. Although these can be discriminated by tissue distribution and some pharmacological features, the P2Y 12 and P2Y 13 receptors form a subgroup of related P2Y subtypes that is structurally different from the other P2Y subtypes but share coupling to G i and a high affinity for ADP.Adenine and uridine nucleotides induce pharmacological and physiological responses through several G-protein-coupled receptors (P2Y) and ligand-gated cation channels (P2X) (1, 2). The P2Y family encompasses two selective purinoceptors: the human P2Y 1 and P2Y 11 receptors, which are preferentially activated respectively by ADP and ATP (3-5). Nucleotide receptors responsive to both adenine and uracil nucleotides are the P2Y 2 receptor, activated equipotently by ATP and UTP (6, 7) as well as the Xenopus P2Y 8 (8) and turkey tp2y receptor (9), activated equally by all triphosphate nucleotides. There are also pyrimidinoceptors: the chicken P2Y 3 (10) and human P2Y 6 (11-13) receptors activated preferentially by UDP, and the human P2Y 4 receptor (13-15) activated preferentially by UTP. All these P2Y subtypes are coupled to the phosphoinositide pathway. The P2Y 11 and tp2y receptors are additionally coupled respectively to stimulation and inhibition of adenylyl cyclase. Other receptors (P2Y 5 , Ref. 16; P2Y 7 , Ref. 17; P2Y 9 , and P2Y 10 ) have been mistakenly included in the P2Y family (18 -20). Recently, a P2Y 12 subtype has been cloned, which corresponds in fact to the platelet ADP receptor previously called P 2T (21,22). It is coupled to an inhibition of adenylyl cyclase and is specifically expressed in the platelets and the brain. Its primary structure is not related to the other P2Y receptors but rather to that of the UDP-glucose receptor (23). Here we report the cloning and characterization of a novel G-protein-coupled receptor that is structurally related to the P2Y 12 receptor and was recently described as an orphan receptor called GPR86 (24).

1 The human P2Y 11 receptor is coupled to both the phosphoinositide and the cyclic AMP pathways. A pharmacological characterization of the recombinant human P2Y 11 receptor has been conducted following stable expression in two di erent cell lines: the 1321N1 astrocytoma cells for inositol trisphosphate measurements and the CHO-K1 cells for cyclic AMP assays. The rank order of potency of a series of nucleotides was almost identical for the two pathways: ATPgS&BzATP4 dATP4ATP4ADPbS42MeSATP. 2 ADPbS, AMPaS and A3P5PS behaved as partial agonists of the human P2Y 11 receptor. At high concentrations, these three nucleotides were able to partially inhibit the ATP response. 3 Suramin was a more potent antagonist than reactive blue 2, whereas pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid was completely inactive. The P2Y 11 receptor proved to be sensitive to suramin in a competitive way with an apparent K i value of 0.82+0.07 mM. 4 The ATP derivative AR-C67085 (2-propylthio-b, g-dichloromethylene-D-ATP), a potent inhibitor of ADP-induced platelet aggregation, was the most potent agonist of the P2Y 11 receptor, among the various nucleotides tested. 5 The pharmacological pro®le of the recombinant human P2Y 11 receptor is closely similar to that of the cyclic AMP-coupled P 2 receptor recently described in HL-60 cells, suggesting that it is the same receptor.