Integrins Regulate the Linkage between Upstream and Downstream Events in G Protein-coupled Receptor Signaling to Mitogen-activated Protein Kinase

Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) can both activate mitogen-activated protein kinase (MAPK), a critical intermediate in the transduction of proliferative signals. Numerous observations have demonstrated that integrin-mediated cell anchorage can regulate the efficiency of signaling from RTKs to MAPK. Recently, a relationship between integrins and GPCR signaling has also emerged; however, little is understood concerning the mechanisms involved. Here, we investigate integrin regulation of GPCR signaling to MAPK, focusing on the P2Y class of GPCRs that function through activation of phospholipase Cbeta. P2Y receptor signaling to the downstream components mitogen-activated protein kinase kinase and MAPK is highly dependent on integrin-mediated cell anchorage. However, activation of upstream events, including inositol phosphate production and generation of calcium transients, is completely independent of cell anchorage. This indicates that integrins regulate the linkage between upstream and downstream events in this GPCR pathway, just as they do in some aspects of RTK signaling. However, the P2Y pathway does not involve cross-activation of a RTK, nor a role for Shc or c-Raf; thus, it is quite distinct from the classical RTK-Ras-Raf-MAPK cascade. Rather, integrin-modulated P2Y receptor stimulation of MAPK depends on calcium and on the activation of protein kinase C

Scale-free foraging by primates emerges from their interaction with a complex environment

Scale-free foraging patterns are widespread among animals. These may be the outcome of an optimal searching strategy to find scarce randomly distributed resources, but a less explored alternative is that this behaviour may result from the interaction of foraging animals with a particular distribution of resources. We introduce a simple foraging model where individuals follow mental maps and choose their displacements according to a maximum efficiency criterion, in a spatially disordered environment containing many trees with a heterogeneous size distribution. We show that a particular tree size frequency distribution induces non-Gaussian movement patterns with multiple spatial scales (L\'evy walks). These results are consistent with tree size variation and Spider monkey (Ateles geoffroyi) foraging patterns. We discuss the consequences that our results may have for the patterns of seed dispersal by foraging primates.Comment: 31 pages, 4 figures. To appear in Proc. Roy. Soc. B. Minor revision

Synthesis and Structure-Activity Relationships of Pyridoxal-6-arylazo-5'-phosphate and Phosphonate Derivatives as P2 Receptor Antagonists.

Novel analogs of the P2 receptor antagonist pyridoxal-5'-phosphate-6-phenylazo-2',4'-disulfonate (PPADS) were synthesized. Modifications were made through functional group substitution on the sulfophenyl ring and at the phosphate moiety through the inclusion of phosphonates, demonstrating that a phosphate linkage is not required for P2 receptor antagonism. Substituted 6-phenylazo and 6-naphthylazo derivatives were also evaluated. Among the 6-phenylazo derivatives, 5'-methyl, ethyl, propyl, vinyl, and allyl phosphonates were included. The compounds were tested as antagonists at turkey erythrocyte and guinea-pig taenia coli P2Y(1) receptors, in guinea-pig vas deferens and bladder P2X(1) receptors, and in ion flux experiments by using recombinant rat P2X(2) receptors expressed in Xenopus oocytes. Competitive binding assay at human P2X(1) receptors in differentiated HL-60 cell membranes was carried out by using [(35)S]ATP-?-S. A 2'-chloro-5'-sulfo analog of PPADS (C(14)H(12)O(9)N(3)ClPSNa), a vinyl phosphonate derivative (C(15)H(12)O(11)N(3)PS(2)Na(3)), and a naphthylazo derivative (C(18)H(14)O(12)N(3)PS(2)Na(2)), were particularly potent in binding to human P2X(1) receptors. The potencies of phosphate derivatives at P2Y(1) receptors were generally similar to PPADS itself, except for the p-carboxyphenylazo phosphate derivative C(15)H(13)O(8)N(3)PNa and its m-chloro analog C(15)H(12)O(8)N(3)ClPNa, which were selective for P2X vs. P2Y(1) receptors. C(15)H(12)O(8)N(3)ClPNa was very potent at rat P2X(2) receptors with an IC(50) value of 0.82 ?M. Among the phosphonate derivatives, [4-formyl-3-hydroxy-2-methyl-6-(2-chloro-5-sulfonylphenylazo)-pyrid-5-yl]methylphosphonic acid (C(14)H(12)-O(8)N(3)ClPSNa) showed high potency at P2Y(1) receptors with an IC(50) of 7.23 ?M. The corresponding 2,5-disulfonylphenyl derivative was nearly inactive at turkey erythrocyte P2Y(1) receptors, whereas at recombinant P2X(2) receptors had an IC(50) value of 1.1 ?M. An ethyl phosphonate derivative (C(15)H(15)O(11)N(3)PS(2)Na(3)), whereas inactive at turkey erythrocyte P2Y(1) receptors, was particularly potent at recombinant P2X(2) receptors

Transformation by a nucleotide-activated P2Y receptor is mediated by activation of Galphai, Galphaq and Rho-dependent signaling pathways

Abstract Background Nucleotide-actived P2Y receptors play critical roles in the growth of tumor cells by regulating cellular proliferation, differentiation and survival. Results Here we demonstrate that an avian P2Y purinoceptor (tP2YR) with unique pharmacological and signal transduction properties induces morphologic and growth transformation of rodent fibroblasts. tP2YR induced a transformed phenotype similar to the mas oncogene, a G protein-coupled receptor which causes transformation by activation of Rac-dependent pathways. tP2YR-transformed cells exhibited increased steady-state activation of Rac1 and RhoA. Like activated Rho GTPases, tP2YR cooperated with activated Raf and caused synergistic transformation of NIH3T3 cells. Our data indicate that the ability of tP2YR to cause transformation is due to its unique ability among purinergic receptors to simultaneously activate Gαq and Gαi. Co-expression of constitutively activated mutants of these two Gα subunits caused the same transformed phenotype as tP2YR and Mas. Furthermore, transformation by both tP2YR and Mas was blocked by pharmacological inhibition of GαI by pertussis toxin (PTX) indicating an essential role for Gαi in transformation by these G-protein coupled receptors. Conclusions Our data suggest that coordinated activation of Gαq and Gαi may link the tP2YR and possibility the Mas oncogene with signaling pathways resulting in activation of Rho family proteins to promote cellular transformation

Molecular cloning, expression and regulatory activity of G α 11 - and βγ -subunit-stimulated phospholipase C- β from avian erythrocytes

A turkey erythrocyte phospholipase C (PLC) has been instrumental in delineating the role of G-proteins in receptor-regulated inositol lipid signalling. This isoenzyme is uniquely regulated both by alpha-subunits of the Gq family and by G-protein beta gamma-subunits. A 4819 bp cDNA encoding this PLC has been cloned from a turkey erythrocyte cDNA library. The open reading frame of this cDNA encodes a 1211-amino-acid protein (calculated molecular mass 139050 Da) that contains amino acid sequences of 16 peptides sequenced from the turkey erythrocyte PLC. The predicted sequence of the turkey PLC shows considerable similarity with the sequences of previously cloned members of the PLC-beta family, with the highest identity (71%) shared with PLC-beta 2 and lesser identities observed with PLC-beta 1 (49%), PLC-beta 3 (46%) and PLC-beta 4 (37%). The largest differences in sequence between the turkey PLC-beta and other PLC-beta isoenzymes occur in the C-terminal domain and in the region between the X- and Y-domains. The turkey isoenzyme and PLC-beta 2, which differ in their regulation by G-protein alpha-subunits, are only 44% similar across the approx. 400 amino acid residues of the C-terminal domain that has been implicated in alpha q activation of these proteins. Recombinant turkey PLC-beta was purified to homogeneity following expression from a recombinant baculovirus in Sf9 insect cells. The immunoreactivity and mobility on SDS/PAGE of the recombinant enzyme were the same as observed with native turkey erythrocyte PLC-beta. Moreover, the catalytic activities of the recombinant enzyme were indistinguishable from those of native turkey erythrocyte PLC-beta in assays carried out in the presence of cholate and Ca2+, or in assays of activity after reconstitution with G alpha 11 or G-protein beta gamma-subunits. The turkey PLC-beta was more sensitive to activation by G alpha 11 than was PLC-beta 2, and was more sensitive to activation by beta gamma-subunits than either PLC-beta 2 or PLC-beta 1

Human P2Y 1 Receptor: Molecular Modeling and Site-Directed Mutagenesis as Tools To Identify Agonist and Antagonist Recognition Sites

The molecular basis for recognition by human P2Y1 receptors of the novel, competitive antagonist 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate (MRS 2179) was probed using site-directed mutagenesis and molecular modeling. The potency of this antagonist was measured in mutant receptors in which key residues in the transmembrane helical domains (TMs) 3, 5, 6, and 7 were replaced by Ala or other amino acids. The capacity of MRS 2179 to block stimulation of phospholipase C promoted by 2-methylthioadenosine 5′-diphosphate (2-MeSADP) was lost in P2Y1 receptors having F226A, K280A, or Q307A mutations, indicating that these residues are critical for the binding of the antagonist molecule. Mutation of the residues His132, Thr222, and Tyr136 had an intermediate effect on the capacity of MRS 2179 to block the P2Y1 receptor. These positions therefore appear to have a modulatory role in recognition of this antagonist. F131A, H277A, T221A, R310K, or S317A mutant receptors exhibited an apparent affinity for MRS 2179 that was similar to that observed with the wild-type receptor. Thus, Phe131, Thr221, His277, and Ser317 are not essential for antagonist recognition. A computer-generated model of the human P2Y1 receptor was built and analyzed to help interpret these results. The model was derived through primary sequence comparison, secondary structure prediction, and three-dimensional homology building, using rhodopsin as a template, and was consistent with data obtained from mutagenesis studies. We have introduced a “cross-docking” procedure to obtain energetically refined 3D structures of the ligand–receptor complexes. Cross-docking simulates the reorganization of the native receptor structure induced by a ligand. A putative nucleotide binding site was localized and used to predict which residues are likely to be in proximity to agonists and antagonists. According to our model TM6 and TM7 are close to the adenine ring, TM3 and TM6 are close to the ribose moiety, and TM3, TM6, and TM7 are near the triphosphate chain

Symplectic connections and Fedosov's quantization on supermanifolds

A (biased and incomplete) review of the status of the theory of symplectic connections on supermanifolds is presented. Also, some comments regarding Fedosov's technique of quantization are made.Comment: Submitted to J. of Phys. Conf. Se

Identification of potent P2Y-purinoceptor agonists that are derivatives of adenosine 5′-monophosphate

1. A series of chain-extended 2-thioether derivatives of adenosine monophosphate were synthesized and tested as agonists for activation of the phospholipase C-linked P2Y-purinoceptor of turkey erythrocyte membranes, the adenylyl cyclase-linked P2Y-purinoceptor of C6 rat glioma cells, and the cloned human P2U-receptor stably expressed in 1321N1 human astrocytoma cells. 2. Although adenosine monophosphate itself was not an agonist in the two P2Y-purinoceptor test systems, eleven different 2-thioether-substituted adenosine monophosphate analogues were full agonists. The most potent of these agonists, 2-hexylthio AMP, exhibited an EC50 value of 0.2 nM for activation of the C6 cell receptor. This potency was 16,000 fold greater than that of ATP and was only 10 fold less than the potency of 2-hexylthio ATP in the same system. 2-hexylthio adenosine was inactive. 3. Monophosphate analogues that were the most potent activators of the C6 cell P2Y-purinoceptor were also the most potent activators of the turkey erythrocyte P2Y-purinoceptor. However, agonists were in general more potent at the C6 cell receptor, and potency differences varied between 10 fold and 300 fold between the two receptors. 4. Although 2-thioether derivatives of adenosine monophosphate were potent P2Y-purinoceptor agonists no effect of these analogues on the human P2U-purinoceptor were observed. 5. These results support the view that a single monophosphate is sufficient and necessary for full agonist activity at P2Y-purinoceptors, and provide insight for strategies for development of novel P2Y-purinoceptor agonists of high potency and selectivity

Structurally related nucleotides as selective agonists and antagonists at P2Y1 receptors

The P2Y1 receptor responds to adenine nucleotides and is present in platelets, heart, smooth muscles prostate, ovary, and brain. A selective antagonist may be useful as an antithrombotic agent. We have analyzed the binding site of this G protein-coupled receptor using ligand design, site-directed mutagenesis, and homology modeling based on rhodopsin. We have designed and synthesized a series of deoxyadenosine 3′,5′-bisphosphate derivatives that act as antagonists, or, in some cases with small structural changes, as agonists or partial agonists. The 2-position accommodates Cl or thioethers, whereas the N6-position is limited to Me or Et. 2′-Substitution with OH or OMe increases agonist efficacy over 2′-H. Using molecular modeling of the binding site, the oxygen atoms of the ribose moiety were predicted to be non-essential, i.e. no specific H-bonds with the receptor protein appear in the model. We have, therefore, substituted this moiety with carbocylics, smaller and larger rings, conformationally constrained rings, and acyclics, with retention of affinity for the receptor. With simplified pharmacophores we are exploring the steric and electronic requirements of the receptor binding site, and the structural basis of receptor activation