Abstract
The B cell antigen receptor (BCR) employs enzymatically inactive adaptor proteins to facilitate activation of intracellular signaling pathways. In animal model systems, adaptor proteins of the growth factor receptor-bound 2 (Grb2) family have been shown to serve critical functions in lymphocytes. However, the roles of Grb2 and the Grb2-related adaptor protein (GRAP) in human B lymphocytes remain unclear. Using TALEN-mediated gene targeting, we show that in human B cells Grb2 and GRAP amplify signaling by the immunoglobulin tail tyrosine (ITT) motif of mIgE-containing BCRs and furthermore connect immunoreceptor tyrosine-based activation motif (ITAM) signaling to activation of the Ras-controlled Erk MAP kinase pathway. In contrast to mouse B cells, BCR-induced activation of Erk in human B cells is largely independent of phospholipase C-ɣ activity and diacylglycerol-responsive members of Ras guanine nucleotide releasing proteins. Together, our results demonstrate that Grb2 family adaptors are critical regulators of ITAM and ITT signaling in naïve and IgE-switched human B cells.
Highlights
Stimulation of the B cell antigen receptor (BCR) activates several intracellular signaling pathways that are executed by a coordinated interplay of several classes of enzymes and catalytically inert adaptor proteins[1]
To this end we generated mutant variants of the human εm immunoglobulin (Ig) heavy chain in which the central immunoglobulin tail tyrosine (ITT) tyrosine residue was replaced by an alanine (YA) or the cytoplasmic tail was shortened to four amino acids (Δtail)
Wild type and mutant εm Ig heavy chains were retrovirally expressed in the human B cell line DG75 and the transfectants were sorted for equal expression of surface mIgE (Fig. 1A)
Summary
Stimulation of the B cell antigen receptor (BCR) activates several intracellular signaling pathways that are executed by a coordinated interplay of several classes of enzymes and catalytically inert adaptor proteins[1]. In mIgG, this immunoglobulin tail tyrosine (ITT) motif was shown to recruit the adaptor protein growth factor receptor-bound 2 (Grb2), which in turn brings Btk directly to the activated mIgG-BCR to facilitate activation of PLC-ɣ2 in memory B cells and promotes production of IgG antibodies in vivo[13,14]. There is an alternative and more ubiquitous mechanism of Ras activation that involves the GEF Sos and its constitutive binding partner Grb[236] In this scenario, which has been well established for receptor tyrosine kinases (RTKs) such as many growth factor receptors, Grb[2] docks with its SH2 domain to conserved tyrosine phosphorylation motifs within the cytoplasmic domains of activated RTKs and brings Sos ‘piggyback’ to the plasma membrane, where it meets and activates membrane-anchored Ras proteins[37]. Which of the two routes leading to activation of Ras and Erk operates in human B cells remains unknown
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