LARD: a new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing

Abstract

Fas and TNF-R1 are cysteine-rich cell surface receptors related to the low-affinity nerve growth factor receptor family. Engagement of these receptors by their respective ligands, FasL and tumor necrosis factor, leads to apoptosis that is signaled through a conserved intracellular portion of the receptor termed the "death domain." We have cloned a new member of this family, lymphocyte-associated receptor of death (LARD), which leads to spontaneous apoptosis when expressed in 293T cells. The expression of LARD is more tightly regulated than that of either Fas or TNF-R1 as it is found predominantly on lymphocytes (T and B cells) but not on macrophages or a number of transformed lymphocyte cell lines. Alternative pre-mRNA splicing generates at least 11 distinct isoforms of LARD. The full-length isoform, LARD-1, extends to include the transmembrane and death domains, whereas the other isoforms encode potentially secreted molecules. Naive B and T cells express very little LARD-1 but express combinations of the other isoforms. Upon T cell activation, a programmed change in alternative splicing occurs so that the full-length, membrane-bound LARD-1 predominates. This may have implications for the control of lymphocyte proliferation following activation.

Figure 1

Alignment of the death domain of LARD with the death domains of other proteins.

Figure 2

Multiple-human-tissue Northern blot probed with LARD cDNA (Upper) and β-actin (Lower). Lanes: 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small intestine; 7, colon; 8, PBL.

Figure 3

RNase protection analysis on lymphocyte subsets. (Upper) LARD expression; (Lower) control protection using a β-actin probe. Lanes: 1, control (no RNA); 2, unstimulated PBL; 3, phytohemagglutinin (PHA)-blasted PBL; 4, CD4⁺ cells; 5, CD8⁺ cells; 6, B cells; 7, macrophages.

Figure 4

Cell death was assessed by trypan blue staining and expressed as a percentage of the total. (A) Cells transfected with CD8 alone or cotransfected with CD8 and LARD. (B) effects of crosslinking with an anti-Fas mAb. F, Fas; L, LARD; F/L, Fas/LARD chimera. (C) Expression of Fas on transfected cells analyzed by a fluorescence-activated cell sorter. These data are representative of three separate experiments.

Figure 5

(A) Schematic representation of the LARD protein drawn above the proposed genomic structure showing the leader peptide, four NGFR repeats, the transmembrane domain, and the death domain. (B) A proposed scheme for the genomic structure of LARD is represented above the prototype LARD-1 cDNA. Alternatively spliced clones LARD-2 through -11 are represented below LARD-1. Positions of the termination codons are represented by an asterisk above the clones. The CAG insertion in clone LARD-1b is indicated above LARD-1.

Figure 6

(A) Southern blots of reverse transcriptase–PCR of LARD cDNA with primers F LARD Kpn and R LARD Xba probed with ³²P-labeled primer F LARD Xba. Lanes: 1, CD4⁺ cells; 2, CD8⁺ cells; 3, B cells; 4, PHA-blasted PBL; 5, negative control. (B) Ethidium-stained control PCR with glyceraldehyde-3-phosphate dehydrogenase primers. Lanes: 1 and 7, φX HaeIII markers; 2, negative control; 3, CD4⁺ cells; 4, CD8⁺ cells; 5, B cells; 6, PHA-blasted peripheral blood mononuclear cells.