Complex chimeras to map ligand binding sites of GPCRs.

Family 1a GPCRs are thought to bind small molecule ligands in a pocket comprising sequences from non-contiguous transmembrane helices. In this study, receptor-ligand binding determinants were defined by building a series of complex chimeras where multiple sequences were exchanged between related G-protein coupled receptors. Regions of P2Y(1), P2Y(2) and BLT(1) predicted to interact with nucleotide and leukotriene ligands were identified and receptors were engineered within their transmembrane helices to transpose the ligand binding site of one receptor on to another receptor. Ligand-induced activation of chimeras was compared with wild-type receptor activation in a yeast reporter gene assay. Binding of ligand to a P2Y(2)/BLT(1) chimera confirmed that the ligand binding determinants of BLT(1) are located in the upper regions of the helices and extracellular loops of this receptor and that they had been successfully transferred to a receptor that normally binds unrelated ligands.

Activity of diadenosine polyphosphates at P2Y receptors stably expressed in 1321N1 cells.

The selectivities of the diadenosine polyphosphates (Ap(n)As, n=2-6) at the human P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(11) receptors stably expressed in 1321N1 human astrocytoma cells was determined using a Fluorescence Imaging Plate Reader (FLIPR) to measure intracellular Ca(2+) mobilisation. The rank order of agonist potencies at P2Y(1) were: ADP>P(1),P(3)-diadenosine triphosphate (Ap(3)A)>P(1),P(3)-diadenosine hexaphosphate (Ap(6)A)=P(1),P(3)-diadenosine diphosphate (Ap(2)A)>>P(1),P(3)-diadenosine pentaphosphate (Ap(5)A). P(1),P(3)-diadenosine tetraphosphate (Ap(4)A) was inactive up to 1 mM. The rank order of agonist potencies at P2Y(2) were: UTP>Ap(4)A>>Ap(6)A>Ap(5)A>Ap(3)A>>Ap(2)A. The Ap(4)A concentration response curve appeared to be bi-phasic. At P2Y(4) all the Ap(n)As tested were inactive as agonists. At P2Y(6), only Ap(3)A and Ap(5)A showed significant agonist activity. At P2Y(11), only Ap(4)A showed significant agonist activity. Ap(n)As were inactive as antagonists of the P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(11) receptors. At P2Y(4), however, the Ap(n)As potentiated the UTP response.

Adenosine nucleotides acting at the human P2Y1 receptor stimulate mitogen-activated protein kinases and induce apoptosis.

For the widely distributed P2Y receptors for nucleotides, the transductional and functional responses downstream of their coupling to G proteins are poorly characterized. Here we describe apoptotic induction and the associated differential stimulation of mitogen-activated protein (MAP) kinase family members by the human P2Y(1) receptor. The potent P2Y(1) receptor agonist, 2-methylthio-ADP (2-MeSADP), stimulated the extracellular-signal regulated kinases (ERK1/2) (EC(50) approximately 5 nm) as well as several, but not all isoforms detected, of the stress-activated protein kinase (SAPK) family. Phospho-isoforms of p38 were unaffected. The induced kinase activity was blocked by the P2Y(1) receptor-selective antagonist, adenosine-2'-phosphate-5'-phosphate, but unaffected by pertussis toxin. In addition, the endogenous ligand ADP, and significantly also 2-MeSATP, induced concentration-dependent phosphorylation changes in the same MAP kinase family members. The sustained activation of ERK1/2 was associated with Elk-1 phosphorylation that was abolished by the MEK1 inhibitor, PD 98059. However, the concomitant transient activation of the SAPKs was not sufficient to induce c-Jun or ATF-2 phosphorylation. The transient phase of the ERK activity was partially inhibited either by the phosphatidylinositol 3-kinase inhibitor, LY 294002, or the PKC inhibitor, Gö 6976. In addition, the Src inhibitor, PP1, or expression of dominant negative Ras also attenuated the transient phase of ERK phosphorylation. In contrast, inhibition of Ras or Src had no effect on the sustained ERK activity, which was critically dependent on phosphatidylinositol 3-kinase. The transient SAPK activity was suppressed by expression of a dominant negative form of MKK4. Furthermore, this kinase-deficient mutant inhibited 2-MeSADP-induced caspase-3 stimulation and the associated decrease in cell number. In conclusion, adenosine di- and triphosphate stimulation of the human P2Y(1) receptor can transiently activate the Ras-ERK cascade via the cooperative effects of phosphatidylinositol 3-kinase, Src and PKC. The sustained ERK stimulation, via a Ras-insensitive pathway, culminates in Elk-1 activation without inducing a proliferation effect. The transient SAPK activity did not evoke transcription factor phosphorylation but was required for the P2Y(1) receptor-mediated apoptotic function.

Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein alpha-subunit chimeras.

The expression of mammalian G protein coupled receptors (GPCRs) in S. cerevisiae provides a powerful assay system for functional analysis, ligand identification and pharmaceutical screening. However, relatively few receptors have been coupled to the pheromone response pathway via the yeast G(alpha), Gpa1p, or chimeric yeast/mammalian G(alpha) subunits containing long C-terminal regions of mammalian G(alpha) proteins. We tested an extended range of seven such chimeras for G(alpha) sub-types of three major classes (G(alphai/o), G(alphas) and G(alphaq)), against eight human GPCRs (SST(2), SST(5), 5-HT(1A), 5-HT(1Dalpha), ML(1B), P2Y(1) and P2Y(2)). Although the G(alphai/o) chimeras increased the range of receptors that coupled efficiently, the G(alphas) and G(alphaq) chimeras were inactive when expressed using the GPA1 promoter. We describe 10 novel Gpa1p chimeras, designated 'transplants', in which the C-terminal five amino acids of Gpa1p were exchanged with mammalian residues. Coupling efficiency and ligand sensitivity improved significantly using the transplants. For the P2Y purinergic receptors, coupling could only be detected with the transplants; this is the first report of G(q) specificity coupling in yeast. Thus, the transplants offer major advantages over previously described approaches, in terms of both the range of receptors coupled and the efficiency of coupling.

Combination of mutations in human immunodeficiency virus type 1 reverse transcriptase required for resistance to the carbocyclic nucleoside 1592U89.

The carbocyclic nucleoside 1592U89 is a selective inhibitor of the human immunodeficiency virus (HIV), targeting the reverse transcriptase (RT). In vitro selection studies were undertaken to generate resistant variants with both HIV type 1 (HIV-1) wild-type strain HIV-1(HXB2) and 3'-azido-3'-deoxythymidine (AZT)-resistant strain HIV-1(RTMC). At least two or three mutations in RT were required to produce a 10-fold reduction in susceptibility. The first RT mutation selected was at codon 184, methionine (M) to valine (V), for HIV-1(HXB2) and HIV-1(RTMC), conferring two- and fivefold resistance, respectively. Two additional mutations were selected with HIV-1(HXB2), either leucine (L) 74 to V and lysine (K) 65 to arginine (R) (first-passage series) or L74 to V and tyrosine (Y) 115 to phenylalanine (F) (second-passage series). Cloned variants, obtained from the 1592U89 selection, were either double RT mutants 65R/184V and 74V/184V or triple RT mutant 74V/115Y/184V. Molecular clones were constructed with single, double, and triple combinations of these mutations for resistance analysis with different RT inhibitors. Each individual mutation conferred only low-level resistance (two- to fourfold) to 1592U89 in the HXB2 background. Double mutants containing the 184V mutation and triple mutants showed slightly greater levels of resistance to 1592U89 (7- to 11-fold). Some of the 1592U89-resistant variants were cross-resistant with 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, and (-)-2'-deoxy-3'-thiacytidine, but none were resistant to 2',3'-didehydro-3'-deoxythymidine or AZT.

Modeling and solution structure probing of the HIV-1 TAR stem-loop.

We present a model for the three-dimensional structure of the HIV TAR stem-loop, based on a modeling algorithm which makes use of the known X-ray coordinates of tRNAs to generate a model structure, which has then been tested experimentally in solution by enzymatic and chemical structure probing of ribo-oligonucleotides encompassing the TAR sequence. The modeling suggested that the structure of TAR was similar to that of the anti-codon loop of tRNA(Asp), having a loop of just three single-stranded residues with a mismatched adenine excluded from the helical stem on the 3' side of the loop. The structural probing is consistent with such a structure for the loop, and reveals an unusual structure around the 5' uridine-rich bulge, which is the binding target for the transactivator protein Tat. These data may be useful in understanding the interaction of TAR with the Tat protein and may aid in the design of anti-AIDS drugs. The coordinates of the model are available on request.

The C-terminal 79 amino acids of the herpes simplex virus regulatory protein, Vmw65, efficiently activate transcription in yeast and mammalian cells in chimeric DNA-binding proteins.

Activation of herpes simplex virus immediate early gene expression normally requires the formation of a ternary complex between a virus trans-activator, Vmw65, a cellular octamer-binding protein, TRF and the cis-acting target sequence, the TAATGARAT motif. We report that the C-terminal 79 amino acids of Vmw65, which contain a potential acidic amphipathic helix, can activate transcription in both yeast and mammalian cells in the absence of TRF interaction when fused to the DNA-binding domain of the yeast transcription factor, GAL4. Together with our previous report which showed that the recruitment of TRF to the DNA by Vmw65 is insufficient for transcription activation, these results indicate that the octamer binding protein may not be directly involved in transcriptional induction mediated by Vmw65. The TRF-Vmw65 complex may therefore represent a novel class of transcription activator in which the protein domain responsible for sequence-specific DNA binding, present in TRF, and that necessary for induction of transcription, within Vmw65, are located on separate proteins. These results are discussed with reference to combinatorial transcriptional control and the role of octamer-binding proteins in other systems.

The amidase regulatory gene (amiR) of Pseudomonas aeruginosa.

Recombinant plasmids carrying the amidase genes of Pseudomonas aeruginosa were used to study the genetic control of amidase synthesis in Escherichia coli and Pseudomonas aeruginosa. The amidase regulator gene, amiR, was found to lie about 2 kbp downstream from the structural gene, amiE. Using plasmids with in vitro-constructed deletions, and plasmids containing subcloned DNA fragments, the amiR gene was located within a 1 kbp ClaI-XhoI DNA fragment. The structural and regulator genes were shown to be transcribed in the same direction. Deletion of DNA sequences between the two genes resulted in increased synthesis of amidase in both E. coli and P. aeruginosa. The intervening sequences showed no repressing effect when tested in trans. The results suggested that the amiR gene could be transcribed from more than one promoter.