The chemokine fractalkine can activate integrins without CX3CR1 through direct binding to a ligand-binding site distinct from the classical RGD-binding site.
The chemokine domain of fractalkine (FKN-CD) binds to the classical RGD-binding site of αvβ3 and that the resulting ternary complex formation (integrin-FKN-CX3CR1) is critical for CX3CR1 signaling and FKN-induced integrin activation. However, only certain cell types express CX3CR1. Here we studied if FKN-CD can activate integrins in the absence of CX3CR1. We describe that WT FKN-CD activated recombinant soluble αvβ3 in cell-free conditions, but the integrin-binding defective mutant of FKN-CD (K36E/R37E) did not. This suggests that FKN-CD can activate αvβ3 in the absence of CX3CR1 through the direct binding of FKN-CD to αvβ3. WT FKN-CD activated αvβ3 on CX3CR1-negative cells (K562 and CHO) but K36E/R37E did not, suggesting that FKN-CD can activate integrin at the cellular levels in a manner similar to that in cell-free conditions. We hypothesized that FKN-CD enhances ligand binding to the classical RGD-binding site (site 1) through binding to a second binding site (site 2) that is distinct from site 1 in αvβ3. To identify the possible second FKN-CD binding site we performed docking simulation of αvβ3-FKN-CD interaction using αvβ3 with a closed inactive conformation as a target. The simulation predicted a potential FKN-CD-binding site in inactive αvβ3 (site 2), which is located at a crevice between αv and β3 on the opposite side of site 1 in the αvβ3 headpiece. We studied if FKN-CD really binds to site 2 using a peptide that is predicted to interact with FKN-CD in site 2. Notably the peptide specifically bound to FKN-CD and effectively suppressed integrin activation by FKN-CD. This suggests that FKN-CD actually binds to site 2, and this leads to integrin activation. We obtained very similar results in α4β1 and α5β1. The FKN binding to site 2 and resulting integrin activation may be a novel mechanism of integrin activation and of FKN signaling.
Department of Dermatology, Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California, United States of America.
Masaaki
M
Fujita
Department of Dermatology, Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California, United States of America.
Yoko K
YK
Takada
Department of Dermatology, Biochemistry and Molecular Medicine, UC Davis School of Medicine, Sacramento, California, United States of America.
Yoshikazu
Y
Takada
D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D016466:CHO Cells; D000073883:CX3C Chemokine Receptor 1; D054428:Chemokine CX3CL1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D039041:Integrin alpha4beta1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D039302:Integrin alphaVbeta3 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020014:K562 Cells; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism*; D019030:Receptors, Vitronectin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020298:U937 Cells
D000073883:CX3C Chemokine Receptor 1; C095235:CX3CR1 protein, human; D054428:Chemokine CX3CL1; D039041:Integrin alpha4beta1; D039302:Integrin alphaVbeta3; D008024:Ligands; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; D019707:Receptors, Chemokine; D019030:Receptors, Vitronectin; C094340:integrin alphavbeta1; C047981:arginyl-glycyl-aspartic acid; D051722:ADAM Proteins; C098660:ADAM15 protein, human