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Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Buhl AM, Pleiman CM, Rickert RC, Cambier JC - J. Exp. Med. (1997)

Bottom Line: PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515.Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses.Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA.

ABSTRACT
Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

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Mutant CD19 (CD19Y484F,Y515F) does not support antigen-induced [Ca2+]i mobilization. J558Lμm3CD45+CD19−, J558Lμm3CD45+  CD19+ and J558Lμm3CD45+CD19+(Y484F,Y515F) cells were loaded with  Indo-1 AM, and analysis of [Ca2+]i initiated before antigen stimulation  (250 ng NP9BSA/106cells/ml). Shown is the mean [Ca2+]i over time with  analysis of ∼600 cells/s. [Ca2+]i in resting cells is 100 nM.
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Figure 8: Mutant CD19 (CD19Y484F,Y515F) does not support antigen-induced [Ca2+]i mobilization. J558Lμm3CD45+CD19−, J558Lμm3CD45+ CD19+ and J558Lμm3CD45+CD19+(Y484F,Y515F) cells were loaded with Indo-1 AM, and analysis of [Ca2+]i initiated before antigen stimulation (250 ng NP9BSA/106cells/ml). Shown is the mean [Ca2+]i over time with analysis of ∼600 cells/s. [Ca2+]i in resting cells is 100 nM.

Mentions: To further explore the requirement of CD19 for PI3-kinase activation and optimal antigen-mediated [Ca2+]i mobilization, we introduced a double-point mutant of CD19, CD19Y484F,Y515F, into the J558Lμm3CD45+ plasmacytoma. The expression levels of sIgM, CD45 and CD19 (Y484F,Y515F) in this cell line is shown in Fig. 1. It has previously been demonstrated that the mutations Y484F and Y515F abrogates the ability of CD19 to interact with the p85-subunit of PI3-kinase (9). Like the CD19-negative cell line, the CD19 mutant cell line, showed no significant antigen-mediated activation of PI3-kinase (results not shown). When we assayed the ability of the cell line expressing the mutant CD19 to mobilize [Ca2+]i in response to antigen stimulation, we found that the mutant CD19 cell line behaved equivalently to the J558Lμm3CD45+ cell line (Fig. 8). This suggests that both CD19-mediated increase in BCR-induced PI3-kinase activation and Ca2+ mobilization require phosphorylation of CD19 Y484 and Y515.


Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization.

Buhl AM, Pleiman CM, Rickert RC, Cambier JC - J. Exp. Med. (1997)

Mutant CD19 (CD19Y484F,Y515F) does not support antigen-induced [Ca2+]i mobilization. J558Lμm3CD45+CD19−, J558Lμm3CD45+  CD19+ and J558Lμm3CD45+CD19+(Y484F,Y515F) cells were loaded with  Indo-1 AM, and analysis of [Ca2+]i initiated before antigen stimulation  (250 ng NP9BSA/106cells/ml). Shown is the mean [Ca2+]i over time with  analysis of ∼600 cells/s. [Ca2+]i in resting cells is 100 nM.
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Related In: Results  -  Collection

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Figure 8: Mutant CD19 (CD19Y484F,Y515F) does not support antigen-induced [Ca2+]i mobilization. J558Lμm3CD45+CD19−, J558Lμm3CD45+ CD19+ and J558Lμm3CD45+CD19+(Y484F,Y515F) cells were loaded with Indo-1 AM, and analysis of [Ca2+]i initiated before antigen stimulation (250 ng NP9BSA/106cells/ml). Shown is the mean [Ca2+]i over time with analysis of ∼600 cells/s. [Ca2+]i in resting cells is 100 nM.
Mentions: To further explore the requirement of CD19 for PI3-kinase activation and optimal antigen-mediated [Ca2+]i mobilization, we introduced a double-point mutant of CD19, CD19Y484F,Y515F, into the J558Lμm3CD45+ plasmacytoma. The expression levels of sIgM, CD45 and CD19 (Y484F,Y515F) in this cell line is shown in Fig. 1. It has previously been demonstrated that the mutations Y484F and Y515F abrogates the ability of CD19 to interact with the p85-subunit of PI3-kinase (9). Like the CD19-negative cell line, the CD19 mutant cell line, showed no significant antigen-mediated activation of PI3-kinase (results not shown). When we assayed the ability of the cell line expressing the mutant CD19 to mobilize [Ca2+]i in response to antigen stimulation, we found that the mutant CD19 cell line behaved equivalently to the J558Lμm3CD45+ cell line (Fig. 8). This suggests that both CD19-mediated increase in BCR-induced PI3-kinase activation and Ca2+ mobilization require phosphorylation of CD19 Y484 and Y515.

Bottom Line: PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515.Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses.Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA.

ABSTRACT
Genetic ablation of the B cell surface glycoprotein CD19 severely impairs the humoral immune response. This requirement is thought to reflect a critical role of CD19 in signal transduction that occurs upon antigen C3dg coligation of antigen receptors with CD19 containing type 2 complement receptors (CR2). Here we show that CD19 plays a key accessory role in B cell antigen receptor signaling independent of CR2 coligation and define molecular circuitry by which this function is mediated. While CD19 is not required for antigen-mediated activation of receptor proximal tyrosines kinases, it is critical for activation of phosphatidylinositol 3-kinase (PI3-kinase). PI3-Kinase activation is dependent on phosphorylation of CD19 Y484 and Y515. Antigen-induced CD19-dependent PI3-kinase activation is required for normal phosphoinositide hydrolysis and Ca2+ mobilization responses. Thus, CD19 functions as a B cell antigen receptor accessory molecule that modifies antigen receptor signaling in a qualitative manner.

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