Abstract
The chemokine receptor CXCR4 is a co-receptor for T-tropic strains of HIV-1. A number of small molecule antagonists of CXCR4 are in development but all are likely to lead to adverse effects due to the physiological function of CXCR4. To prevent these complications, allosteric agonists may be therapeutically useful as adjuvant therapy in combination with small molecule antagonists. A synthetic cDNA library coding for 160,000 different SDF-based peptides was screened for CXCR4 agonist activity in a yeast strain expressing a functional receptor. Peptides that activated CXCR4 in an autocrine manner induced colony formation. Two peptides, designated RSVM and ASLW, were identified as novel agonists that are insensitive to the CXCR4 antagonist AMD3100. In chemotaxis assays using the acute lymphoblastic leukemia cell line CCRF-CEM, RSVM behaves as a partial agonist and ASLW as a superagonist. The superagonist activity of ASLW may be related to its inability to induce receptor internalization. In CCRF-CEM cells, the two peptides are also not inhibited by another CXCR4 antagonist, T140, or the neutralizing monoclonal antibodies 12G5 and 44717.111. These results suggest that alternative agonist-binding sites are present on CXCR4 that could be screened to develop molecules for therapeutic use.
Highlights
CXCR4 is a member of the chemokine receptor family of G protein-coupled receptors
Larger molecules bind to specific sites on the extracellular surface of the receptor leading to a conformational change that is transmitted to an intracellular G␣␥ complex
Allosteric agonists or modulators of CXCR4 activity would be therapeutically useful but have not yet been identified. Such agents could preserve the function of CXCR4 and minimize adverse effects expected from the use of competitive receptor antagonists in HIV-1 therapy
Summary
CXCR4 is a member of the chemokine receptor family of G protein-coupled receptors. A number of mechanisms for receptor activation of G protein-coupled receptors have been proposed [1, 2]. Larger molecules bind to specific sites on the extracellular surface of the receptor leading to a conformational change that is transmitted to an intracellular G␣␥ complex This results in an exchange of GTP for GDP in the G␣ protein, dissociation of G␣ from the G␥ complex, and activation of downstream signal transduction pathways. Allosteric modulators that bind to different sites on G protein-coupled receptors should have no effect on activity except in the presence of receptor agonists or antagonists [3]. A two-step process has been proposed involving the binding of a chemokine to its receptor followed by placement of the N-terminal flexible loop of the chemokine on another site of the receptor, leading to its activation In this model, the N-loop, consisting of the first 20 –30 residues of chemokines, provides most of the specificity for the receptor. We focus on the characterization of two peptide agonists, designated RSVM and ASLW, that are resistant to the small molecule CXCR4 antagonists AMD3100 and T140 and the neutralizing antibodies
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