Aberrant Trafficking of the B Cell Receptor Ig-αβ Subunit in a B Lymphoma Cell Line

Abstract

The B cell Ag receptor (BCR) has two important functions: first, it binds and takes up Ag for presentation to T lymphocytes; and second, it transmits signals that regulate B cell development. Normal expression of the BCR requires the association of the Ag binding subunit, membrane IgM (mIgM), with the signaling component, the Ig-αβ heterodimer. After assembly in the endoplasmic reticulum, the intact BCR travels through the secretory pathway to the cell surface. In this paper, we report two variants of the B lymphoma cell lines, WEHI 279 and WEHI 231, that have both lost the ability to express μ heavy chain and consequently do not express mIgM. However, these variants do express the Ig-αβ heterodimer. In one variant, WEHI 279*, the Ig-αβ remained trapped intracellularly in the absence of mIgM. The other variant, 303.1.5.LM, expressed an aberrantly glycosylated Ig-αβ on the cell surface that was capable of signaling after cross-linking with anti-Ig-β Abs. Further characterization uncovered a point mutation in the 303.1.5.LM mb1 gene that would change a proline for a leucine in the extracellular domain of Ig-α. The 303.1.5.LM Ig-αβ could not associate with a wild-type mIgM after μ heavy chain was reconstituted by DNA transfection. Thus, this mutation could define a region of the Ig-α polypeptide that is important for recognition by the endoplasmic reticulum quality control system, for association with glycosylating enzymes, and for the association of Ig-αβ subunits with mIgM subunits to create a complete BCR complex.

Localization in membrane microdomains of the Ig-α/β component of the BCR expressed by a B lymphoma variant

“…For anti--induced BCR cross-linking, 2 ml cells (2.5 × 10 7 ) were stimulated with 100 g of goat anti-mouse heavy chain Abs. For anti-Ig-␤-induced clustering of the BCR or Ig-␣/␤, the 2 ml cells (2.5 × 10 7 ) were stimulated with 600 g of biotinylated HM79-16 mAb followed 2 min later with 80 g of streptavidin [11]. Immediately following stimulation, 5 ml of ice cold PBS containing 1 mM sodium orthovanadate was added and the cells were pelleted at 800 rpm at 4 • C for 5 min.…”

“…Cells were solubilized at 4 • C in MG lysis buffer (20 mM Tris-HCl pH 8.0, 1% Triton X-100, 2 mM EDTA, 10% glycerol, 137 mM NaCl, 1 M pepstatin A, 1 M aprotinin, 1 M PMSF and 1 M leupeptin) [11] or in CSB buffer (20 mM Hepes, pH 7.6, 0.5% Triton X-100, 10.3% sucrose, 20 mM NaCl, 5 mM MgC l2 , 0.1 mg/ml BSA, 2 mM sodium orthovanadate, 2.5 mM phenylmethylsulfonyl fluoride (PMSF), 500 g/ml aprotinin, 500 g/ml leupeptin and 0.8 g/ml pepstatin) [6,15]. After lysis, the cell extracts were chilled on ice for 10 min and then the nuclei and insoluble material were removed by centrifuging at 14,000 rpm for 10 min in a cold microfuge.…”

“…The rabbit anti-mouse Ig-␣ cytoplasmic domain Ab, rabbit anti-mouse Ig-␤ cytoplasmic domain Ab, rabbit anti-mouse Ig-␤ extracellular domain Ab and mouse monoclonal anti-phosphotyrosine Abs (4G10) have been described previously [11][12][13]. Anti-Akt and anti-phospho-Akt (serine 473) Ab were from Cell Signaling Technology (Mississauga, Ont., Canada).…”

“…DeFranco (University of California, San Francisco, CA). The WEHI 231 variant, WEHI 303.1.5LM was described previously [10,11]. Cells were grown at 37 • C in RPMI 1640 supplemented with heat inactivated 10% FCS (Intergen, Purchase, NY), 50 M 2-␤-ME, 500 U/ml penicillin, 500 g/ml streptomycin, and 2 mM l-glutamine.…”

“…To test whether Ig-␣/␤ contains the structural information that allows for lipid raft translocation, we examined the ability of the Ig-␣/␤ portion of the BCR, expressed by itself on the cell surface, to localize into lipid rafts after antibody-mediated cross-linking. Our approach was to use the mIgM-deficient variant of the WEHI 231 B cell line, WEHI 303.1.5 that expresses Ig-␣/␤ on the cell surface without mIgM, similar to the expression patterns of pro-B celllike cell lines [9][10][11]. Although the Ig-␣/␤ on WEHI 303.1.5 was able to enter lipid rafts after being cross-linked, this was less efficient than translocation of the intact BCR complex.…”