SummaryAlthough there is a mounting body of evidence that eosinophils are recruited to sites of allergic inflammation by a number of ~-chemokines, particularly eotaxin and RANTES, the receptor that mediates these actions has not been identified. We have now cloned a G protein-coupled receptor, CC CKR3, from human eosinophils which, when stably expressed in AML14.3D10 cells bound eotaxin, MCP-3 and R_ANTES with Kas of 0.1, 2.7, and 3.1 nM, respectively. CC CKR3 also bound MCP-1 with lower affinity, but did not bind MIP-10~ or MIP-I[~. Eotaxin, RANTES, and to a lessor extent MCP-3, but not the other chemokines, activated CC CKR3 as determined by their ability to stimulate a CaZ+-flux. Competition binding studies on primary eosinophils gave binding at~inities for the different chemokines which were indistinguishable from those measured with CC CKR3. Since CC CKR3 is prominently expressed in eosinophils we conclude that CC CKR3 is the eosinophil eotaxin receptor. Eosinophils also express a much lower level of a second chemokine receptor, CC CKR1, which appears to be responsible for the effects of MIP-llx.

The guanine nucleotide-binding proteincoupled receptor superfamily binds a vast array of biological messengers including lipids, odorants, catecholamines, peptides, and proteins. While some small molecules bind to these receptors at a single interhelical site, we find that the binding domain on the receptor for the inflammatory protein C5a is more complex and consists of two distinct subsites. This more elaborate motif appears to be an evolutionary adaptation of the simpler paradigm to which a second interaction site has been added in the receptor N terminus. Surprisingly, occupation of only one of the subsites is required for receptor activation. The two-site motif is not unique to the C5a receptor but appears to be widely used by the superfamily to accommodate macromolecular ligands.The 74-aa glycoprotein C5a evokes a variety of responses in vivo and in vitro, implying that it is a principal mediator of inflammatory responses (1, 2). C5a is a potent chemotaxin and secretagogue for granulocytes and macrophages; it activates the respiratory burst in these cells and modulates their adhesive properties. The effects of C5a are amplified by its ability to stimulate the release of other mediators including histamine, prostaglandins, leukotrienes, interleukin (IL) 1, and IL-6 (1-3).All of the effects of C5a are initiated when it binds to its cell surface receptor, a member ofthe guanine nucleotide-binding protein (G protein)-coupled receptor superfamily (4, 5). The superfamily consists of over 100 members and binds a variety of ligands ranging in complexity from small molecules to moderately sized proteins. Despite this biologic diversity, a general model for the structure of these receptors has emerged: an extracellular N terminus, seven membranespanning helices connected by alternating intracellular and extracellular loops, and an intracellular C terminus (6, 7). The amino acid sequence of the C5a receptor is consistent with this model and like most members of the family has a short N terminus of about 30 residues in length (4, 5).Family members such as rhodopsin and the ,3adrenergic receptor bind their ligands at a single domain, which lies in the receptor's hydrophobic core, between the helices and below the upper plane of the cellular membrane (6, 8). However, it is unclear whether this binding motif is also used by other members of the superfamily, especially those that interact with more complex ligands like C5a, or whether the motif is altered to accommodate the larger agonists. The little information that exists comes largely from studies with the glycopeptide hormone receptors, a branch ofthe superfamily characterized by a greatly extended extracellular N terminus (9, 10). These receptors, in contrast to rhodopsin and the ,fadrenergic receptor, appear to bind ligands by means of this enlarged N terminus (11,12). We now report that the binding site of the C5a receptor is more complex and consists of two physically separable domains. The first domain is composed of the N terminus and possibly the exter...

The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.Human immunodeficiency virus (HIV) therapies have been highly successful in slowing disease progression, increasing health and well-being, and prolonging life. However, viral resistance is now becoming common, and since most existing drugs target only two viral proteins, reverse transcriptase and protease, cross-resistance is a significant problem. One solution to the issue of resistance is development of new complementary therapies based on novel mechanisms of action. The discovery that the chemokine receptors CCR5 and CXCR4, in addition to CD4, are required for viral entry not only furthered understanding of the fusion and infection process but provided two new targets for therapeutic intervention (3,12,14,17,18,22,44).The entry mechanism as currently understood is an ordered process in which the viral envelope protein, gp120, following interaction with CD4, undergoes a conformational change allowing binding to the appropriate chemokine receptor, CCR5 for macrophagetropic or R5 strains, and CXCR4 for T-celltropic or X4 s...

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Human CCR5 is a G-coupled receptor that binds to the envelope protein gp120 and CD4 and mediates the HIV-1 viral entry into the cells. The blockade of this binding by a small molecule receptor antagonist could lead to a new mode of action agent for HIV-1 and AIDS. Screening of natural product extracts led to the identification of anibamine (1), a novel pyridine quaternary alkaloid as a TFA salt, from Aniba sp.; ophiobolin C from fermentation extracts of fungi Mollisia sp.; and 19,20-epoxycytochalasin Q from Xylaria sp. Formation of the TFA salt of anibamine is plausibly an artifact of the isolation. The identity of the natural counterion is unknown. Anibamine.TFA competed for the binding of 125I-gp120 to human CCR5 with an IC50 of 1 microM. Ophiobolin C and 19,20-epoxycytochalasin Q exhibited binding IC50) values of 40 and 60 microM, respectively.