RESUMO
The surface-expressed transmembrane CX3C chemokine ligand 1 (CX3CL1/fractalkine) induces firm adhesion of leukocytes expressing its receptor CX3CR1. After shedding by the disintegrins and metalloproteinases (ADAM) 10 and 17, CX3CL1 also acts as soluble leukocyte chemoattractant. Here, we demonstrate that transmembrane CX3CL1 expressed on both endothelial and epithelial cells induces leukocyte transmigration. To investigate the underlying mechanism, we generated CX3CR1 variants lacking the intracellular aspartate-arginine-tyrosine (DRY) motif or the intracellular C-terminus which led to a defect in intracellular calcium response and impaired ligand uptake, respectively. While both variants effectively mediated firm cell adhesion, they failed to induce transmigration and rather mediated retention of leukocytes on the CX3CL1-expressing cell layer. Targeting of ADAM10 led to increased adhesion but reduced transmigration in response to transmembrane CX3CL1, while transmigration towards soluble CX3CL1 was not affected. Thus, transmembrane CX3CL1 mediates leukocyte transmigration via the DRY motif and C-terminus of CX3CR1 and the activity of ADAM10.
Assuntos
Quimiocina CX3CL1/metabolismo , Leucócitos/fisiologia , Migração Transendotelial e Transepitelial/fisiologia , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Receptor 1 de Quimiocina CX3C , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Células Cultivadas , Quimiocina CX3CL1/genética , Quimiotaxia/fisiologia , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Humanos , Leucócitos/citologia , Ligantes , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Receptores de Quimiocinas/química , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismoRESUMO
The transmembrane chemokine CX3CL1 is expressed on the endothelial surface and promotes leukocyte adhesion and transmigration by receptor interaction via its extracellular chemokine domain. Since little is known about its intracellular C-terminus, we examined the consequences of C-terminal truncation on cellular distribution, proteolytic shedding and function of murine CX3CL1. Full length murine CX3CL1 was expressed and shed by the metalloproteinase ADAM10 as described for human CX3CL1. Truncation of murine CX3CL1 led to reduced maturation and impaired trafficking to the surface. Truncation of CX3CL1 also abrogated localization to early endosomal vesicles, but increased shedding from the surface by ADAM10. Once truncated CX3CL1 was expressed on the surface, it mediated cell contact and induced leukocyte transmigration similar as full length CX3CL1. These data suggest that the C-terminus of CX3CL1 carries important determinants for cellular trafficking but not for function of the chemokine during leukocyte recruitment.