Tyrosine phosphorylation of scavenger receptor cysteine‐rich WC1 is required for the WC1‐mediated potentiation of TCR‐induced T‐cell proliferation

Abstract

Workshop cluster 1 (WC1) molecules are transmembrane glycoproteins uniquely expressed by cd T cells. They belong to the scavenger receptor cysteine-rich superfamily and are encoded by a multi-gene family, which is divided on the basis of antibody reactivity, into three groups, WC1.1, WC1.2, and WC1.3. The potential role of WC1 as a co-stimulatory molecule for the cd TCR is suggested by the presence of several tyrosinebased motifs in their intracellular domains. In this study, we found that WC1 was constitutively phosphorylated in ex vivo bovine cd T cells and associated with src family tyrosine kinases. Crosslinking of WC1 molecules resulted in an increase in WC1 phosphorylation and co-crosslinking of WC1 and cd TCR together prolonged WC1 phosphorylation. We identified the second tyrosine residue as the primary phosphorylation target in WC1.1 and WC1.2 intracellular sequences in both in vitro and in vivo assays. The cytoplasmic tails of WC1.1 and WC1.2 were phosphorylated on serine and PKC activity was required for PMA-induced endocytosis of WC1.1 or WC1.2. We found that phosphorylation of the second tyrosine in the WC1 cytoplasmic domain was required for the WC1-mediated potentiation of TCR-induced T-cell proliferation, suggesting that WC1 acts as a co-stimulatory molecule for cd TCR.Key words: Bovine . Cell surface molecules . gd T cells . Signal transduction IntroductionIn adult chickens, ruminants and pigs, unlike in mice and humans, gd T cells can constitute 30% of total PBMC [1]. A majority of these gd T cells in ruminants bear the glycoprotein lineage marker Workshop cluster 1 (WC1) on their surface. Based on the profiles of their gene expression, WC1 1 gd T cells of cattle represent the inflammatory population, whereas WC1 À gd T cells are regulatory cells [2,3]. WC1, a member of the scavenger receptor cysteine-rich (SRCR) superfamily, is categorized into group B along with CD5, CD6, and CD163, based on the organization of cysteine residues in its extracellular SRCR domains [4]. Like the SRCR family member SpSRCR, which is expressed in the immune cells of the purple sea urchin [5], WC1 molecules are products of a large gene family, and thus have the potential to increase the diversity of immune responses independently of the adaptive immune response receptor, i.e. the TCR.Bovine gd T cells express at least three known variants, WC1.1, WC1.2, and WC1.3, which are defined by antibodies recognizing unknown epitopes on their SRCR domains [6]. WC1.1 and WC1.2 are expressed on two mostly non-overlapping subsets of bovine gd T cells, and WC1.3 is expressed on a small 254subpopulation of WC1.1 1 cells. Cytokine production and cellular proliferation in response to stimulation vary according to the forms of WC1 expressed by gd T cells [7]. Ex vivo WC1.1 1 gd T cells, but not WC1.2 1 gd T cells, proliferate well to the gd T-cell antigens of Leptospira, and produce IFN-g in response to either antigen or 8].The correlation of the expression of a variable WC1 gene product with a cellular immune response to antigen su...

WC1 Is a Hybrid γδ TCR Coreceptor and Pattern Recognition Receptor for Pathogenic Bacteria

“…We have previously shown that the gd T cell specific WC1 transmembrane proteins, members of the ancient and conserved scavenger receptor cysteine rich (SRCR) receptor superfamily closely related to CD163 molecules (17), can act as coreceptors for gd T cells. Ab-mediated crosslinking of WC1 with the TCR-CD3 complex potentiates T cell activation, whereas ligation of WC1 alone has no effect or is inhibitory (18,19). This potentiation of TCR signaling requires the phosphorylation of a tyrosine in the cytoplasmic tails of WC1 (19,20) and is modulated by an endocytic dileucine motif and membrane proximal serines (21).…”

“…Ab-mediated crosslinking of WC1 with the TCR-CD3 complex potentiates T cell activation, whereas ligation of WC1 alone has no effect or is inhibitory (18,19). This potentiation of TCR signaling requires the phosphorylation of a tyrosine in the cytoplasmic tails of WC1 (19,20) and is modulated by an endocytic dileucine motif and membrane proximal serines (21). Following activation, WC1 colocalizes with the gd TCR-CD3 complex in lipid rafts (22).…”

The Endocytosis and Signaling of the γδ T Cell Coreceptor WC1 Are Regulated by a Dileucine Motif

“…Coligation of WC1 with TCR/CD3 transduces a positive signal (21,22), which is dependent upon the phosphorylation of a tyrosine in the WC1 cytoplasmic domain (22,23). Each WC1 molecule has 6-11 extracellular group B scavenger receptor cysteine-rich domains of ∼100 aa, each containing three or four disulfide bonds and a hydrophobic core.…”

“…The cytoplasmic domains of WC1 proteins are similar to those of CD3g, CD3d, and CD4, containing two consensus [DE]XXXL[LIM] dileucine motifs, multiple serines, and two to four YXXØ tyrosine-based motifs (20,23). We showed previously that mutation of the tyrosine of one of the YXXØ tyrosine-based motifs in WC1-3 does not affect phorbol ester-induced endocytosis, suggesting that the dileucine motifs may instead regulate WC1 phorbol esterinduced endocytosis (22). In this study, we investigate the requirement for two serines and the membrane proximal dileucine motif in phorbol ester-induced WC1 endocytosis in both HEK-293T cells and Jurkat T cells.…”

Adhesion Molecules and the Cellular Population of the Normal Camel (Camelus dromedaries) Mammary Glands

“…Study with wide range of monoclonal antibodies revealed that WC+1 + corecptor is made of tow nonoverlaping variants, WC+1.1 and WC+1.2, whereas WC+1.3 variant is coexpressed with the WC+1.1 [20]. WC+1 + coreceptors play major role in mediating the costimulatory signal in activation of T cells [21]. Further studies on the role of the WC+1 + have revealed a diverse genetic variation which could reach to at least 13 WC+1 + genes on the bovine chromosome 5 [22].…”