(A) A20IIA1.6 cells were surface-biotinylated with Sulfo-NHS-SS-Biotin on ice, stimulated with anti-IgG for the indicated times (min) and then biotin was stripped from the cell surface. Lysed samples were precipitated sequentially with streptavidin and then anti-Igα antibodies. Parallel samples were then immunoblotted with anti-phosphotyrosine (4G10) or anti-Igα antibodies. (B) Cells were labeled and stimulated as above except that biotin was not stripped from the cell surface. Streptavidin precipitations were then sequentially immunoblotted with 4G10 and anti-Igα antibodies. (C) Resting surface-biotinylated cells were directly lysed and sequentially precipitated with streptavidin and then anti-Igα antibodies. Precipitations were Western blotted with anti-Igα antibodies.

Aliquots of wild-type A20IIA1.6 cells were surface-biotinylated and then stimulated through the BCR for the indicated times. Following lysis, cells were precipitated with strepavidin, 4G10, or anti-Igα antibodies. Precipitations were resolved by SDS-PAGE, transferred to nylon membrane and immunoblotted sequentially with 4G10 (A) and then anti-Igα antibodies (B). Results of a typical experiment are presented ( n = 3). Arrows denote Igα.

(A) Schematic representation of the exclusive and independent models of BCR phosphorylation and internalization. The independent model includes all the reactions in the exclusive model plus the additional ones indicated. Eliminating the sequestration reaction corresponds to clathrin deletion while eliminating the removal reaction corresponds to dn-dynamin (see discussion). (B) Extent of phosphorylation for wild-type (red), dn-dynamin (green), and clathrin mutant scenarios (blue). (C) Maximal signaling through the BCR, as assayed by Igα phosphorylation, as a function of ligand avidity. A comparison of exclusive (red) and independent (green) signaling and internalization models is provided (inset).
Time to maximum BCR signal intensity as a function of ligand avidity as predicted for the exclusive (red) and independent (green) models. The parameters used to generate the figure are k _off = 2.0 min ⁻¹, k _+a = k _–a = 4.0 min ⁻¹, k _+s = 8.0 min ⁻¹, k _–s = 4.0 min ⁻¹, k _p = 10.0 min ⁻¹, k _r = 5.0 min ⁻¹, k _c = 4.0 min ⁻¹, k _d = 2.0 min ⁻¹, and k _g = 2.0 min ⁻¹; K _i = K _M = 0.1 in units such that the initial BCR concentration is unity. The rate parameters were selected to give for the wild-type a maximum phosphorylation of about 10% at 2–3 min and decay on the order of 10 min. In the case of the non-exclusive model, we substitute either k _p = 12.0 min ⁻¹ (faster phosphorylation), k _c = 2.0 min ⁻¹ (slower sequester) or k _d = 8.0 min ⁻¹ with k _g = 4.0 min ⁻¹ (faster removal) to maintain the same maximum integrated phosphorylation to facilitate comparison. Data for scaled k _d and k _g are shown; scaled k _p and k _c give qualitatively similar behavior.

A20IIA1.6 cells expressing chimeras containing the cytosolic tail of either wild-type Igα or Igα in which the ITAM or non-ITAM tyrosines were mutated were assayed for endocytosis. (A) Comparison of Igα ^wt (solid squares) to Igα in which ITAM tyrosines were mutated to phenylalanines (Igα ^YΔF182,193) (open triangles) or alanines (Igα ^YΔA182,193) (open squares). (B) Comparison of the internalization of Igα ^wt (solid squares), a cytosolic tail truncation of Igα (ΔT, open triangles), and a ITAM/non-ITAM Igα mutant (Igα ^{YΔA176,182,193,204}, open squares).
(C and D) Igα ^wt (C) or Igα ^{YΔA176,182,193,204} (D) were assayed for endocytosis in the presence (closed symbols) or absence of Latrunculin A (open symbols).

(A) A20IIA1.6 cells expressing PDGFR chimeric receptors containing the cytosolic tail of either Igα or Igα ^YΔF204 were stimulated through the chimera with FITC-conjugated antibodies for 10 min, fixed, permeabilized, and counterstained with PE-BLNK-SH2 (SH2). Cells were then visualized using confocal microscopy. (B) BLNK ^kd cells were stimulated with Cy5-conjugated anti-IgG antibodies for 10 min. Samples were then fixed, permeabilized, counterstained with PE-BLNK-SH2, and visualized by confocal microscopy. Typical results are shown ( n = 150 cells from three independent experiments). (C) Purified BLNK ^−/− splenic B cells were stimulated with FITC-conjugated anti-IgM F(ab) ₂ fragments for 10 or 15 min and then fixed, permeabilized, counterstained with PE-BLNK-SH2, and visualized by confocal microscopy. Small arrowheads indicate internalized non-phosphorylated BCR complexes while large arrowheads indicate locations of phosphorylated surface BCRs.

In (A–C), A20IIA1.6 wild-type- or dn-dynamin-expressing cells were stimulated with anti-IgG for the indicated times (min). (A) Total cell lysates were immunoblotted with 4G10. (B) BLNK, Igα, or PLCγ2 immunoprecipitates were resolved by SDS-PAGE and then immunoblotted as indicated. (C) Total cell lysates were immunoblotted with phospho-specific antibodies to Erk, Jnk, or p38. Membranes were then stripped and reprobed with antibodies against Erk, Jnk, or p38 as indicated. (D) Cells were stimulated with decreasing amounts of anti-IgG (shown in μg/ml) for 3 min. Cells were then assayed for Erk activation as in (C).