RESUMO
Chemokine-receptor interactions regulate leukocyte trafficking during inflammation. CC chemokines exist in equilibrium between monomeric and dimeric forms. Although the monomers can activate chemokine receptors, dimerization is required for leukocyte recruitment in vivo, and it remains controversial whether dimeric CC chemokines can bind and activate their receptors. We have developed an obligate dimeric mutant of the chemokine monocyte chemoattractant protein-1 (MCP-1) by substituting Thr(10) at the dimer interface with Cys. Biophysical analysis showed that MCP-1(T10C) forms a covalent dimer with similar structure to the wild type MCP-1 dimer. Initial cell-based assays indicated that MCP-1(T10C) could activate chemokine receptor CCR2 with potency reduced 1 to 2 orders of magnitude relative to wild type MCP-1. However, analysis of size exclusion chromatography fractions demonstrated that the observed activity was due to a small proportion of MCP-1(T10C) being monomeric and highly potent, whereas the majority dimeric form could neither bind nor activate CCR2 at concentrations up to 1 µM. These observations help to reconcile previous conflicting results and indicate that dimeric CC chemokines do not bind to their receptors with affinities approaching those of the corresponding monomeric chemokines.
Assuntos
Quimiocina CCL2/metabolismo , Mutação de Sentido Incorreto , Multimerização Proteica , Substituição de Aminoácidos , Linhagem Celular , Quimiocina CCL2/química , Quimiocina CCL2/genética , Humanos , Receptores CCR2/química , Receptores CCR2/genética , Receptores CCR2/metabolismoRESUMO
The transmembrane proteins Delta and Notch act as ligand and receptor in a conserved signaling pathway required for a variety of cell fate specification events in many organisms. Binding of Delta to Notch results in a proteolytic cascade that releases the Notch intracellular domain, allowing it to participate in transcriptional activation in the nucleus. Recent research has implicated the endocytic and ubiquitylation machinery as essential components of Delta-Notch signaling. Our analysis of chimeric and missense Delta variants has delineated a number of structural requirements for Delta trafficking, receptor binding, and signaling. We find that while the Delta N-terminal domain is necessary and sufficient for binding to Notch, the integrity of the epidermal-growth-factor-like repeat (ELR) 2 is also required for Notch binding. Screening of 117 Delta mutant lines for proteins that exhibit aberrant subcellular trafficking has led to the identification of 18 Delta alleles (DlTD alleles) that encode "trafficking-defective" Delta proteins. We find, unexpectedly, that many DlTD alleles contain missense mutations in ELRs within the Delta extracellular domain. Finally, we find that two DlTD alleles contain lysine missense mutations within the Delta intracellular domain (DeltaICD) that may identify residues important for DeltaICD mono-ubiquitylation and subsequent Delta endocytosis and signaling.
Assuntos
Drosophila melanogaster/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Receptores Notch/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Endocitose , Feminino , Glicosilação , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Receptores Notch/genética , Homologia de Sequência de Aminoácidos , Frações SubcelularesRESUMO
To identify the elements of two chemokines [monocyte chemoattractant protein-1 (MCP-1) and eotaxin] that control their differential recognition by their respective receptors (CCR2 and CCR3), we have studied the receptor interactions of MCP-1-eotaxin chimeras. Each receptor was found to exhibit a distinct binding preference for proteins containing the amino-terminal region of the cognate chemokine for that receptor. However, other elements dictating chemokine preference were different for the two receptors. In some cases, the influence of replacing a particular region was dependent on the identities of neighboring regions, indicating a complex network of cooperative and/or compensating interactions.
Assuntos
Quimiocina CCL2/metabolismo , Quimiocinas CC/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Quimiocina CCL11 , Quimiocina CCL2/química , Quimiocina CCL2/genética , Quimiocinas/farmacologia , Quimiocinas CC/química , Quimiocinas CC/genética , Clonagem Molecular , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Conformação Proteica , Receptores de Quimiocinas/efeitos dos fármacos , Receptores de Quimiocinas/fisiologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sensibilidade e EspecificidadeRESUMO
The specificity of leukocyte trafficking in inflammation is controlled by the interactions of chemokines with chemokine receptors. Reliable structure-function studies of chemokine-receptor interactions would benefit from cell lines that express consistent high levels of chemokine receptors. We describe herein two new Chinese hamster ovary (CHO) cell lines in which the genes for chemokine receptors CCR2 and CCR3 have been incorporated into identical positions in the host genome. CCR2 is the primary receptor for the chemokine monocyte chemoattractant protein-1 (MCP-1) whereas CCR3 is the primary receptor for the chemokines eotaxin-1, eotaxin-2 and eotaxin-3. Both receptors are expressed at >5,000,000 copies per cell, substantially higher levels than in previous cell lines, and both are competent for binding and activation by the cognate chemokines for these receptors. Using these cell lines we confirm that eotaxin-1 and eotaxin-3 can act as an agonist and an antagonist, respectively, of CCR2. In addition, we show that eotaxin-2 is an antagonist of CCR2 and MCP-1 is an agonist of CCR3. Comparison of the chemokine sequences reveals several positions that are identical in MCP-1 and eotaxin-1 but different in eotaxin-2 and eotaxin-3, suggesting that these amino acids play a role in CCR2 activation.
