title: The CXC chemokine receptor 4 ligands ubiquitin and stromal cell-derived factor-1α function through distinct receptor interactions.
abstract: Recently, we identified extracellular ubiquitin as an endogenous CXC chemokine receptor (CXCR) 4 agonist. However, the receptor selectivity and molecular basis of the CXCR4 agonist activity of ubiquitin are unknown, and functional consequences of CXCR4 activation with ubiquitin are poorly defined. Here, we provide evidence that ubiquitin and the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α do not share CXCR7 as a receptor. We further demonstrate that ubiquitin does not utilize the typical two-site binding mechanism of chemokine-receptor interactions, in which the receptor N terminus is important for ligand binding. CXCR4 activation with ubiquitin and SDF-1α lead to similar Gα(i)-responses and to a comparable magnitude of phosphorylation of ERK-1/2, p90 ribosomal S6 kinase-l and Akt, although phosphorylations occur more transiently after activation with ubiquitin. Despite the similarity of signal transduction events after activation of CXCR4 with both ligands, ubiquitin possesses weaker chemotactic activity than SDF-lα in cell migration assays and does not interfere with productive entry of HIV-1 into P4.R5 multinuclear activation of galactosidase indicator cells. Unlike SDF-1α, ubiquitin lacks interactions with an N-terminal CXCR4 peptide in NMR spectroscopy experiments. Binding and signaling studies in the presence of antibodies against the N terminus and extracellular loops 2/3 of CXCR4 confirm that the ubiquitin CXCR4 interaction is independent of the N-terminal receptor domain, whereas blockade of extracellular loops 2/3 prevents receptor binding and activation. Our findings define ubiquitin as a CXCR4 agonist, which does not interfere with productive cellular entry of HIV-1, and provide new mechanistic insights into interactions between CXCR4 and its natural ligands.
authors.affiliation: Department of Surgery, Burn and Shock Trauma Institute, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois 60153, USA.
authors.forename: Vikas
authors.initials: V
authors.lastname: Saini
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Staren
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Ziarek
authors.forename: Zayd N
authors.initials: ZN
authors.lastname: Nashaat
authors.forename: Edward M
authors.initials: EM
authors.lastname: Campbell
authors.forename: Brian F
authors.initials: BF
authors.lastname: Volkman
authors.forename: Adriano
authors.initials: A
authors.lastname: Marchese
authors.forename: Matthias
authors.initials: M
authors.lastname: Majetschak
mesh_terms: D002460:Cell Line; D054377:Chemokine CXCL12 / Q000378:metabolism* / Q000494:pharmacology; D002633:Chemotaxis / Q000187:drug effects; D015658:HIV Infections / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008024:Ligands; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D019718:Receptors, CXCR4 / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D054377:Chemokine CXCL12; D008024:Ligands; D019718:Receptors, CXCR4; D025801:Ubiquitin; D020928:Mitogen-Activated Protein Kinases