The anti-HIV cytokine midkine binds the cell surface-expressed nucleolin as a low affinity receptor.
The growth factor midkine (MK) is a cytokine that inhibits the attachment of human immunodeficiency virus particles by a mechanism similar to the nucleolin binding HB-19 pseudopeptide. Here we show that the binding of MK to cells occurs specifically at a high and a low affinity binding site. HB-19 prevents the binding of MK to the low affinity binding site only. Confocal immunofluorescence laser microscopy revealed the colocalization of MK and the cell-surface-expressed nucleolin at distinct spots. The use of various deletion constructs of nucleolin then indicated that the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, is the domain that binds MK. The specific binding of MK to cells is independent of heparan sulfate and chondroitin sulfate expression. After binding to cells, MK enters cells by an active process. Interestingly, the cross-linking of surface-bound MK with a specific antibody results in the clustering of surface nucleolin along with glycosylphosphatidylinositol-linked proteins CD90 and CD59, thus, pointing out that MK binding induces lateral assemblies of nucleolin with specific membrane components of lipid rafts. Our results suggest that the cell surface-expressed nucleolin serves as a low affinity receptor for MK and could be implicated in its entry process.
Said
Elias A
EA
Unité de Virologie et Immunologie Cellulaire (URA 1930 CNRS), Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Krust
Bernard
B
Nisole
Sébastien
S
Svab
Josette
J
Briand
Jean-Paul
JP
Hovanessian
Ara G
AG
D000818:Animals; D019380:Anti-HIV Agents / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D016207:Cytokines / Q000378:metabolism*; D017930:Genes, Reporter; D015658:HIV Infections / Q000517:prevention & control; D015497:HIV-1 / Q000472:pathogenicity* / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism*; D000078792:Midkine; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017978:Receptors, Growth Factor / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013601:T-Lymphocytes; D014162:Transfection; D000097796:Nucleolin
D019380:Anti-HIV Agents; D002352:Carrier Proteins; D016207:Cytokines; D008562:Membrane Glycoproteins; D000078792:Midkine; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011956:Receptors, Cell Surface; D017978:Receptors, Growth Factor; D011994:Recombinant Proteins; D000097796:Nucleolin; C408049:midkine receptors