title: Photolabeling identifies transmembrane domain 4 of CXCR4 as a T140 binding site.
abstract: CXCR4, a G-protein-coupled receptor, which binds the chemokine stromal cell-derived factor 1 alpha (SDF-1alpha, CXCL12), is one of two co-receptors most frequently used by HIV-1 to infect CD4+ lymphocytes. The SDF-1alpha/CXCR4 axis is also involved in angiogenesis, in stem cell homing to bone marrow, in rheumatoid arthritis and in cancer. Here, we directly determined the binding site of the inverse agonist T140 on CXCR4 using photoaffinity labeling. Two T140 photoanalogs were synthesized containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in positions 5 or 10, yielding [Bpa(5)]T140 and [Bpa(10)]T140. Binding experiments on HEK293 cells stably expressing the wild-type CXCR4 receptor using 125I-SDF-1alpha demonstrated that T140 and both photoanalogs had affinities in the nanomolar range, similar to SDF-1alpha. Photolabeling led to the formation of specific, covalent 42 kDa T140-CXCR4 complexes. V8 protease digestion of both CXCR4/125I-[Bpa(5)]T140 and CXCR4/125I-[Bpa(10)]T140 adducts generated a fragment of 6kDa suggesting that the T140 photoanalogs labeled a fragment corresponding to Lys(154)-Glu(179) of the receptor's 4th transmembrane domain. Further digestion of this 6kDa fragment with endo Asp-N led to the generation of a shorter fragment validating the photolabeled region. Our results demonstrate that T140 interacts with residues of the fourth transmembrane domain of the CXCR4 receptor and provide new structural constraints enabling us to model the complex between T140 and CXCR4.
authors.affiliation: Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Philip E
authors.initials: PE
authors.lastname: Boulais
authors.forename: Dominic
authors.initials: D
authors.lastname: Dulude
authors.forename: Jérôme
authors.initials: J
authors.lastname: Cabana
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Heveker
authors.forename: Emanuel
authors.initials: E
authors.lastname: Escher
authors.forename: Pierre
authors.initials: P
authors.lastname: Lavigne
authors.forename: Richard
authors.initials: R
authors.lastname: Leduc
mesh_terms: D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001577:Benzophenones / Q000737:chemistry*; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D007457:Iodine Radioisotopes; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D019920:Photoaffinity Labels; D017434:Protein Structure, Tertiary; D011533:Proviruses / Q000235:genetics; D019718:Receptors, CXCR4 / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: C488060:4-benzoylphenylalanine; D019380:Anti-HIV Agents; D001577:Benzophenones; C527276:CXCR4 protein, human; D004279:DNA, Viral; D007457:Iodine Radioisotopes; D009842:Oligopeptides; D019920:Photoaffinity Labels; D019718:Receptors, CXCR4; D010649:Phenylalanine; C116977:T140 peptide