To determine the function of immunoglobulin (Ig)α immunoreceptor tyrosine–based activation motif (ITAM) phosphorylation, we generated mice in which Igα ITAM tyrosines were replaced by phenylalanines (IgαFF/FF). IgαFF/FFmice had a specific reduction of B1 and marginal zone B cells, whereas B2 cell development appeared to be normal, except that λ1 light chain usage was increased. The mutants responded less efficiently to T cell–dependent antigens, whereas T cell–independent responses were unaffected. Upon B cell receptor ligation, the cells exhibited heightened calcium flux, weaker Lyn and Syk tyrosine phosphorylation, and phosphorylation of Igα non-ITAM tyrosines. Strikingly, when the Igα ITAM mutation was combined with a truncation of Igβ, B cell development was completely blocked at the pro-B cell stage, indicating a crucial role of ITAM phosphorylation in B cell development.

The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)α and Igβ, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Igβ, we produced mice that carry a deletion of the cytoplasmic domain of Igβ (IgβΔC mice) and compared them to mice that carry a similar mutation in Igα (MB1ΔC, herein referred to as IgαΔC mice). IgβΔC mice differ from IgαΔC mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca2+ flux. However, IgβΔC B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Igβ is required for B cell development beyond the immature B cell stage and that Igα and Igβ have distinct biologic activities in vivo.

Antigen receptor genes are assembled through a mechanism known as V(D)J recombination, which involves two different joining reactions: signal and coding joining. Formation of these joints is essential for antigen receptor assembly as well as maintaining chromosomal integrity. Here we report on a cell-free system for coding joint formation using deletion and inversion recombination substrates. In vitro coding joint formation requires RAG1, RAG2, and heat-labile factors present in the nuclear extract of nonlymphoid cells. Both inversion-and deletion-mediated coding joint reactions produce diverse coding joints, with deletions and P nucleotide addition. We also show that deletion-mediated coding joint formation follows the 12/23 rule and requires the catalytic subunit of DNA-dependent protein kinase.B and T lymphocytes produce diverse antigen receptors utilizing the V(D)J recombination reaction. The DNA sequence requirements for V(D)J recombination consist of highly conserved heptamer and nonamer DNA motifs (recombination signal sequences [RSSs]) separated by a spacer of 12 or 23 bp (12 RSS and 23 RSS), which flank recombining regions of both the immunoglobulin and T-cell receptor loci. Efficient recombination occurs almost exclusively between RSSs with different spacer lengths. This restriction, known as the 12/23 rule (25,49), has been shown to be regulated during the cleavage step of V(D)J recombination (7,46,52). Orientation of the RSSs that are to be recombined (head-to-head or head-to-tail orientation) determines whether rearrangement follows a deletional or inversional mechanism (25).The first step of the V(D)J recombination reaction involves specific recognition and cleavage at the RSSs by RAG1 and RAG2 (15,28,50). Competition assays have suggested that the nonamer motif may play a fundamental role in sequence-specific recognition (4, 34), and direct binding of RAG1 to the nonamer motif has been demonstrated (5, 44). Interestingly, this binding activity was mapped to a domain which shows significant homology to the DNA binding domain of the Hin family of bacterial invertases (5, 44). A stable complex of RAG1 and RAG2 with a RSS has recently been isolated, suggesting that RAG1 and RAG2 may function as a complex during V(D)J recombination. This conclusion is supported by immunoprecipitation studies (23,45).Cleavage occurs at the heptamer/coding border in a characteristic manner. First, RAG1 and/or RAG2 introduce a nick at the heptamer/coding border, followed by a nucleophilic attack by the free hydroxyl group on the bottom strand of DNA, resulting in the generation of a double-strand break at the signal end and a hairpin at the coding end (28). These two types of ends are generated through a one-step transesterification reaction similar to that used by both Mu transposase and human immunodeficiency virus integrase (51). The hairpin coding ends produced are joined imprecisely to form a coding joint, and the blunt 5Ј phosphorylated signal ends are also joined (in a precise manner) to form signal joints. The im...