title: Small molecule chemokine mimetics suggest a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3.
abstract: OBJECTIVE
  The chemokine receptor CXCR3 directs migration of T-cells in response to the ligands CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. Both ligands and receptors are implicated in the pathogenesis of inflammatory disorders, including atherosclerosis and rheumatoid arthritis. Here, we describe the molecular mechanism by which two synthetic small molecule agonists activate CXCR3.
  
  
  METHODS
  As both small molecules are basic, we hypothesized that they formed electrostatic interactions with acidic residues within CXCR3. Nine point mutants of CXCR3 were generated in which an acidic residue was mutated to its amide counterpart. Following transient expression, the ability of the constructs to bind and signal in response to natural and synthetic ligands was examined.
  
  
  RESULTS
  The CXCR3 mutants D112N, D195N and E196Q were efficiently expressed and responsive in chemotaxis assays to CXCL11 but not to CXCL10 or to either of the synthetic agonists, confirmed with radioligand binding assays. Molecular modelling of both CXCL10 and CXCR3 suggests that the small molecule agonists mimic a region of the '30s loop' (residues 30-40 of CXCL10) which interacts with the intrahelical CXCR3 residue D112, leading to receptor activation. D195 and E196 are located in the second extracellular loop and form putative intramolecular salt bridges required for a CXCR3 conformation that recognizes CXCL10. In contrast, CXCL11 recognition by CXCR3 is largely independent of these residues.
  
  
  CONCLUSIONS
  We provide here a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Such findings may have implications for the design of CXCR3 antagonists.
authors: Leukocyte Biology Section, NHLI Division, Faculty of Medicine, Imperial College London, UK.
authors: Belinda
authors: B
authors: Nedjai
authors: Hubert
authors: H
authors: Li
authors: Ilana L
authors: IL
authors: Stroke
authors: Emma L
authors: EL
authors: Wise
authors: Maria L
authors: ML
authors: Webb
authors: J Robert
authors: JR
authors: Merritt
authors: Ian
authors: I
authors: Henderson
authors: Anthony E
authors: AE
authors: Klon
authors: Andrew G
authors: AG
authors: Cole
authors: Richard
authors: R
authors: Horuk
authors: Nagarajan
authors: N
authors: Vaidehi
authors: James E
authors: JE
authors: Pease
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D054357:Chemokine CXCL10 / Q000378:metabolism*; D054371:Chemokine CXCL11 / Q000378:metabolism*; D002633:Chemotaxis / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D054448:Precursor Cells, B-Lymphoid / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay; D054367:Receptors, CXCR3 / Q000819:agonists* / Q000235:genetics; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D014162:Transfection
chemical_list: C114753:CXCL10 protein, human; C515202:CXCL11 protein, human; C515187:CXCR3 protein, human; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D018076:DNA, Complementary; D008024:Ligands; D054367:Receptors, CXCR3; D054852:Small Molecule Libraries; D000242:Cyclic AMP