title: Binding of cytoplasmic proteins to the CD19 intracellular domain is high affinity, competitive, and multimeric.
abstract: CD19 is required for the development of B1 and marginal zone B cells, for Ab responses, and for B cell memory. CD19 immunoprecipitates contain a complex of cytoplasmic proteins, including Lyn, Vav, phospholipase Cgamma2 (PLCgamma2), Grb2, and the p85 subunit of phosphatidylinositol 3-kinase. Which of these bind directly to CD19 and the strengths of the interactions are unknown. These issues are important in understanding the signaling functions of CD19, which are crucial for normal B cell physiology. Using purified, recombinant proteins, we now show that each of these signaling proteins contains at least one Src homology 2 (SH2) domain that interacts directly with the phosphorylated CD19 cytoplasmic domain. The affinities of binding of the SH2 domains of Vav, p85, and Grb2 to CD19 are each in the nanomolar range by surface plasmon resonance (Biacore) analysis. Binding of Lyn and PLCgamma2 do not fit 1:1 modeling. However, analyses of binding data (Lyn) and competition experiments (PLCgamma2) suggest that these bind with comparable affinity. Competition experiments demonstrate that SH2 domains whose binding is dependent on the same CD19 tyrosine(s) compete for binding, but these SH2 domains do not impede binding of different SH2 domains to other CD19 tyrosines. We conclude that binding to the CD19 cytoplasmic domain is multimeric, high affinity, and competitive. The high affinity of the interactions also suggests that tyrosines that were nonessential in vivo are nevertheless functional. A preliminary structural model suggests that CD19 forms a signaling complex containing multiple cytoplasmic proteins in close proximity to each other and to the plasma membrane.
authors.affiliation: Department of Microbiology, University of Alabama, 701 South 19th Street, Birmingham, AL 35294, USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Brooks
authors.forename: Perry M
authors.initials: PM
authors.lastname: Kirkham
authors.forename: Larry
authors.initials: L
authors.lastname: Freeberg
authors.forename: Robert H
authors.initials: RH
authors.lastname: Carter
mesh_terms: D018941:Antigens, CD19 / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000737:chemistry*; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D020349:Surface Plasmon Resonance; D014443:Tyrosine; D018909:src Homology Domains
chemical_list: D018941:Antigens, CD19; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine