Genome of the bacterium Streptococcus pneumoniae strain R6.

Streptococcus pneumoniae is among the most significant causes of bacterial disease in humans. Here we report the 2,038,615-bp genomic sequence of the gram-positive bacterium S. pneumoniae R6. Because the R6 strain is avirulent and, more importantly, because it is readily transformed with DNA from homologous species and many heterologous species, it is the principal platform for investigation of the biology of this important pathogen. It is also used as a primary vehicle for genomics-based development of antibiotics for gram-positive bacteria. In our analysis of the genome, we identified a large number of new uncharacterized genes predicted to encode proteins that either reside on the surface of the cell or are secreted. Among those proteins there may be new targets for vaccine and antibiotic development.

Cloning of CCRL1, an orphan seven transmembrane receptor related to chemokine receptors, expressed abundantly in the heart.

In an attempt to identify novel chemokine receptor genes, cDNA expressed sequence tags (EST) were analyzed for a significant homology with mammalian chemokine receptors. The sequence from one of the selected EST clones was used to generate a full-length cDNA encoding a putative seven transmembrane receptor, CCRL1-CC chemokine receptor like 1. The full-length receptor encodes a polypeptide of 350 amino acids and has about 35% homology to the chemokine receptors CCR6 and CCR7. Northern blot analysis indicates predominant expression of about 5.0, 2.0 and 1.3kb mRNA forms in human heart tissue, while low-level expression of the 2.0 and 1.3kb forms was observed in lung, pancreas and spleen and in fetal tissues. In-situ hybridization confirmed the presence of CCRL1 mRNA in cardiac muscle cells. Similar to the chemokine receptor CCR6, CCRL1 maps to chromosome 6 and has one intron in the 5' untranslated region. Coupled transcription-translation of CCRL1 cDNA yielded a glycosylated polypeptide of about 45kDa.

High-throughput screening for ligand-induced c-fos mRNA expression by branched DNA assay in Chinese hamster ovary cells.

We have developed a generally useful screening assay for receptor agonists and antagonists in Chinese Hamster Ovary (CHO) cells. Three key features of the assay make it applicable to a broad range of receptors: (1) the use of CHO cells as host cells to overexpress receptors, (2) measurement of endogenous c-fos mRNA, which responds to a wide spectrum of stimuli, and (3) the use of branched chain DNA assay which is highly sensitive, quantifiable, amenable to high-throughput analysis, and easy to execute. The combination of these features provides a powerful means to screen rapidly for peptide and small molecule ligands for a variety of receptors. CHO cells overexpressing insulin receptor were used as a test system to compare conventional signaling assays with the high-throughput c-fos branched DNA assay.

Interleukin-8 receptors R1 and R2 activate mitogen-activated protein kinases and induce c-fos, independent of Ras and Raf-1 in Chinese hamster ovary cells.

Many of the biological effects of interleukin-8 (IL-8) are realized by binding to the two seven-transmembrane receptors IL-8 R1 and IL-8 R2. IL-8 R1 is activated only by IL-8, while IL-8 R2 is activated by IL-8, GROalpha, and a few other alpha chemokines. In addition to the well-known chemoattractant function, IL-8 is also angiogenic and mitogenic. IL-8 R1 and R2 have been shown to interact with Galphai2 and Galpha16, resulting in the activation of several mitogen-activated protein kinases. We have investigated IL-8 R1 and IL-8 R2 regulated upstream mediators and downstream effects of extracellularly responsive kinase (ERK) signaling pathways by expressing the individual receptors in a heterologous system. Our results demonstrate the following in CHO cells stably expressing either IL-8 R1 or R2 receptors: (a) IL-8 activates ERK and ERK kinases (MEK) through R1. Both IL-8 and GROalpha activate ERK and MEK through R2, whereas MIP-1alpha, a beta chemokine, does not activate these kinases through either of these receptors. (b) ERK activation is inhibited by pertussis toxin and MEK1 inhibitor. (c) ERK activation is independent of the upstream mediators Ras and Raf-1. (d) The downstream effects of ERK activation result in an increase of c-fos mRNA through both R1 and R2 receptors.

Inhibition of adenylyl cyclase by alpha chemokines IL-8 and GRO-alpha in Chinese hamster ovary cells expressing R1 and R2 receptors.

Interleukin-8 (IL-8) and growth-related oncogene protein-alpha (GRO-alpha) belong to a family of alpha chemokines. The biologic effects of IL-8 are realized by binding to two seven-transmembrane receptors R1 and R2 and that of GRO-alpha by binding to receptor R2. Chinese hamster ovary (CHO) cells stably expressing R1 and R2 have been used to demonstrate that the ligand-dependent signaling by both receptors is via the inhibition of adenylyl cyclase. This inhibition is pertussis toxin sensitive and could be mediated by G(alpha)i2, which is present in CHO cells. GRO-alpha inhibits adenylyl cyclase exclusively in CHO-R2 cells, and IL-8 inhibits in both CHO-R1 and CHO-R2 cells. The cAMP status in cells is an easy, reliable, quantifiable signal that is amenable to high throughput screening for small molecule analogs.

Receptor recognition and specificity of interleukin-8 is determined by residues that cluster near a surface-accessible hydrophobic pocket.

To determine the regions of interleukin-8 (IL-8) that allow high affinity and interleukin-8 receptor type 1 (IL8R1)-specific binding of chemokines, we produced chimeric proteins containing structural domains from IL-8, which binds to both IL8R1 and interleukin-8 receptor type 2 (IL8R2) with high affinity, and from GRO gamma, which does not bind to IL8R1 and binds to IL8R2 with reduced affinity. Receptor binding activity was tested by competition of 125I-IL-8 binding to recombinant IL8R1 and IL8R2 cell lines. Substitution into IL-8 of the GRO gamma sequences corresponding to either the amino-terminal loop (amino acids 1-18) or the first beta-sheet (amino acids 18-32) reduced binding to both IL8R1 and IL8R2. The third beta-sheet of IL-8 (amino acids 46-53) was required for binding to IL8R1 but not IL8R2. Exchanges of the second beta-sheet (amino acids 32-46) or the carboxyl-terminal alpha-helix (amino acids 53-72) had no significant effect. When IL-8 sequences were substituted into GRO gamma, a single domain containing the second beta-sheet of IL-8 (amino acids 18-32) was sufficient to confer high affinity binding for both IL8R1 and IL8R2. The amino-terminal loop (amino acids 1-18) and the third beta-sheet (amino acids 46-53) of IL-8 had little effect when substituted individually but showed increased binding to both receptors when substituted in combination. Individual amino acid substitutions were made at positions where IL-8 and GRO gamma sequences differ within the regions of residues 11-21 and 46-53. IL-8 mutations L49A or L49F selectively inhibited binding to IL8R1. Mutations Y13L and F21N enhanced binding to IL8R1 with little effect on IL8R2. A combined mutation Y13L/S14Q selectively decreased binding to IL8R2. Residues Tyr13, Ser14, Phe21, and Lys49 are clustered in and around a surface-accessible hydrophobic pocket on IL-8 that is physically distant from the previously identified ELR binding sequence. A homology model of GRO gamma, constructed from the known structure of IL-8 by refinement calculations, indicated that access to the hydrophobic pocket was effectively abolished in GRO gamma. These studies suggest that the surface hydrophobic pocket and/or adjacent residues participate in IL-8 receptor recognition for both IL8R1 and IL8R2 and that the hydrophobic pocket itself may be essential for IL8R1 binding. Thus this region contains a second site for IL-8 receptor recognition that, in combination with the Glu4-Leu5-Arg6 region, can modulate receptor binding affinity and IL8R1 specificity.