The Juxtamembrane Domain of Butyrophilin BTN3A1 Controls Phosphoantigen-Mediated Activation of Human Vγ9Vδ2 T Cells.

Vγ9Vδ2 T lymphocytes are the major human peripheral γδ T cell subset, with broad reactivity against stressed human cells, including tumor cells. Vγ9Vδ2 T cells are specifically activated by small phosphorylated metabolites called phosphoantigens (PAg). Stress-induced changes in target cell PAg levels are specifically detected by butyrophilin (BTN)3A1, using its intracellular B30.2 domain. This leads to the activation of Vγ9Vδ2 T cells. In this study, we show that changes in the juxtamembrane domain of BTN3A1, but not its transmembrane domain, induce a markedly enhanced or reduced γδ T cell reactivity. There is thus a specific requirement for BTN3A1's juxtamembrane domain for correct γδ T cell-related function. This work identified, as being of particular importance, a juxtamembrane domain region of BTN3A molecules identified as a possible dimerization interface and that is located close to the start of the B30.2 domain.

The intracellular B30.2 domain of butyrophilin 3A1 binds phosphoantigens to mediate activation of human Vγ9Vδ2 T cells.

In humans, Vγ9Vδ2 T cells detect tumor cells and microbial infections, including Mycobacterium tuberculosis, through recognition of small pyrophosphate containing organic molecules known as phosphoantigens (pAgs). Key to pAg-mediated activation of Vγ9Vδ2 T cells is the butyrophilin 3A1 (BTN3A1) protein that contains an intracellular B30.2 domain critical to pAg reactivity. Here, we have demonstrated through structural, biophysical, and functional approaches that the intracellular B30.2 domain of BTN3A1 directly binds pAg through a positively charged surface pocket. Charge reversal of pocket residues abrogates binding and Vγ9Vδ2 T cell activation. We have also identified a gain-of-function mutation within this pocket that, when introduced into the B30.2 domain of the nonstimulatory BTN3A3 isoform, transfers pAg binding ability and Vγ9Vδ2 T cell activation. These studies demonstrate that internal sensing of changes in pAg metabolite concentrations by BTN3A1 molecules is a critical step in Vγ9Vδ2 T cell detection of infection and tumorigenesis.

Competition of PTB with TIA proteins for binding to a U-rich cis-element determines tissue-specific splicing of the myosin phosphatase targeting subunit 1.

A considerable amount of smooth muscle phenotypic diversity is generated by tissue-specific and developmentally regulated splicing of alternative exons. The control mechanisms are unknown. We are using a myosin phosphatase targeting subunit-1 (MYPT1) alternative exon as a model to investigate this question. In the present study, we show that the RNA binding proteins TIA and PTB function as antagonistic enhancers and suppressors of splicing of the alternative exon, respectively. Each functions through a single U-rich element, containing two UCUU motifs, just downstream of the alternative exon 5' splice site. Tissue-specific down-regulation of TIA protein in the perinatal period allows PTB to bind to the U-rich element and suppress splicing of the alternative exon as the visceral smooth muscle acquires the fast-phasic smooth muscle contractile phenotype. This provides a novel role for PTB in the tissue-specific regulation of splicing of alternative exons during the generation of smooth muscle phenotypic diversity.

The CD44 alternative v9 exon contains a splicing enhancer responsive to the SR proteins 9G8, ASF/SF2, and SRp20.

The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering approximately 30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing.