Association of BAP31 with CD11b/CD18. Potential role in intracellular trafficking of CD11b/CD18 in neutrophils.
The beta2 integrin CD11b/CD18 is an integral membrane protein that is present in the plasma membrane and secondary granules of neutrophils and functions as a major adhesion molecule. Upon cellular activation, there is translocation of intracellular pools of CD11b/CD18 to the plasma membrane in concert with enhanced cellular adhesion. Although much is known about the function of CD11b/CD18, how this protein is transported within the cell is less well defined. Here we report that CD11b/CD18 specifically binds to BAP31, a member of a novel class of sorting proteins regulating cellular anterograde transport. Through experiments aimed at identifying CD11b/CD18-binding proteins, we produced a monoclonal antibody termed E1B2 that recognizes a 28-kDa membrane protein that co-precipitates with CD11b/CD18. Microsequence analysis of the E1B2 antigen revealed that it is BAP31. Co-association of CD11b/CD18 and BAP31 was confirmed in co-immunoprecipitation and protein binding assays. Additional experiments revealed that the binding of BAP31 to CD11b/CD18 was not dependent on divalent cations nor mediated by the I-domain of CD11b. Using glutathione S-transferase fusion chimeras, we determined that binding of CD11b/CD18 to BAP31 is mediated through interactions with the cytoplasmic tail of BAP31. Immunolocalization studies revealed colocalization of BAP31 and CD11b/CD18 within neutrophil secondary granules. Subcellular fractionation studies in polymorphonuclear leukocytes (PMN) revealed similar patterns of redistribution of BAP31 and CD11b/CD18 from fractions enriched in secondary granules to the plasma membrane following stimulation with formylmethionylleucylphenylalanine (fMLP). Given the known sorting properties of BAP31, these findings suggest that BAP31 may play a role in regulating intracellular trafficking of CD11b/CD18 in neutrophils.
Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, USA. kaen@emory.edu
Ke
K
Zen
Markus
M
Utech
Yuan
Y
Liu
Illena
I
Soto
Asma
A
Nusrat
Charles A
CA
Parkos
D000595:Amino Acid Sequence; D001692:Biological Transport; D039481:CD11b Antigen / Q000737:chemistry*; D018821:CD18 Antigens / Q000737:chemistry*; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005982:Glutathione Transferase / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism
C091247:BCAP31 protein, human; D039481:CD11b Antigen; D018821:CD18 Antigens; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D005982:Glutathione Transferase