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

Abstract

WC1 proteins, which are specifically expressed by bovine γδ T cells from a gene array containing 13 members, are part of the scavenger receptor cysteine-rich family. WC1 cytoplasmic domains contains multiple tyrosines, one of which is required to be phosphorylated for TCR coreceptor activity, and a dileucine endocytosis motif. Like the TCR coreceptor CD4, WC1 is endocytosed in response to PMA. Because WC1 endocytosis may play a role in the activation of γδ T cells, we examined WC1 endocytosis in the adherent cell 293T and Jurkat T cell lines using a fusion protein of extracellular CD4 and the transmembrane and cytoplasmic domain of WC1. Individual mutation of the two leucine residues of the endocytic dileucine motif in the WC1 cytoplasmic domain significantly reduced PMA-induced endocytosis in both cell types and enhanced IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1 in Jurkat cells, suggesting that the sustained membrane coligation of CD3/TCR with WC1 caused by a decrease in endocytosis increases T cell activation. Mutation of two serines upstream of the endocytic dileucine motif affected endocytosis only in adherent 293T cells. Although the two upstream serines were not required for WC1 endocytosis in Jurkat cells, the pan–protein kinase C inhibitor Gö6983 blocked endocytosis of CD4/WC1, and mutation of the upstream serines in WC1 inhibited IL-2 production stimulated by cocross-linking of CD3/TCR and CD4/WC1. These studies provide insights into the signaling of WC1 gene arrays that are present in most mammals and play critical roles in γδ T cell responses to bacterial pathogens.

Variegated Transcription of the WC1 Hybrid PRR/Co-Receptor Genes by Individual γδ T Cells and Correlation With Pathogen Responsiveness

“…Our previous studies support a model of γδ T cell activation in which pathogen ligation of the WC1 co-receptors along with the TCR is necessary for optimal cellular responses ( 32 , 47 ). Moreover, crosslinking of the γδ TCR with WC1s using an mAb that recognizes most WC1 molecules (mAb CC15) has been shown to give a stronger and more amplified signal compared with crosslinking with an mAb that recognizes a specific WC1 molecule within the family of 13 (i.e., WC1-8 by mAb CACT21A) ( 24 ).…”

“…Based on these observations, we sought to determine if the WC1 gene transcription was different among clones that responded to Leptospira (WC1.1 cohort) compared with those that generally did not (WC1.2 cohort) since we have shown previously that 5 of the 13 bovine WC1 molecules have SRCR domains that directly bind Leptospira (i.e., WC1-3, WC1-6, WC1-8, WC1-10, and WC1-13) ( 23 ). Since antibody-mediated co-ligation of WC1 and TCR/CD3 results in augmented proliferation and IFNγ production, it suggests that pathogen-mediated co-ligation of WC1 and TCR/CD3 would also and thus that WC1 expression and pathogen binding specificity would influence γδ T cell responsiveness ( 24 , 25 , 32 ). 80% of the WC1.1 cohort of clones that were generated with cells from a primed animal and continually cultured with Leptospira had transcripts for at least one WC1 gene that had a binding domain for Leptospira (Figure 5 ).…”

Bovine gamma delta T cells and the function of gamma delta T cell specific WC1 co-receptors

“…Phosphorylated YDDV in type III WC1 cytoplasmic domains would be more likely to bind the adaptor proteins SLP-76 or Nck [86,87]. Mutation of two membrane proximal serines in the type I WC1 cytoplasmic domain, one of which is in a consensus protein kinase C (PKC) phosphorylation motif, also significantly decreased WC1 coreceptor activity [88]. cytoplasmic domain significantly decreases PMA-induced endocytosis.…”

“…Interestingly, coligation of WC1 cytoplasmic domains with TCR/CD3 results in differential levels of IL-2 secretion: type I < type II < type III. This can be correlated with differences in the nucleation of the signaling complex downstream of WC1 [68] but it may also be due to differences in the two endocytic dileucine motifs in type II and III cytoplasmic domains that would be predicted to decrease clathrin-mediated endocytosis and prolong signaling [88].…”

“…Serines (SS or SD), a functional dileucine motif (LL) or mutated motifs (LI or II), and tyrosine motifs are indicated. Phosphorylated tyrosines are indicated by a circled P[67][68][69]88].…”