Monstrated no obvious co-localisation with CD63. Collectively these data suggest that degradation of CTLA-4 is related to its endocytic capacity and that the presence of YV/ EKM motif promotes both internalisation and sensitivity to lysosomal degradation. Moreover, trout CTLA-4 contains an alternate tyrosine-based motif that has a reduced efficiency of endocytosis and has a more stable cell surface MedChemExpress I-BRD9 phenotype when compared to CTLA-4 from human, chicken and xenopus.is seen as an increase in the Alexa647 signal 1676428 over time. Notably, human CTLA-4 and chimeric chicken CTLA-4 (YVKM containing proteins) showed a comparable rate of recycling (Figure 5A and B) whereas chimeric xenopus and trout CTLA-4, which lacked YVKM motif were notably less efficient. Since an acidic residue (E) at the +1 position of YxxM has been suggested to favour an interaction with the lysosomal sorting adaptor AP-3 [17], we mutated the YVKM motif in human CTLA-4 to YEKM and assayed the efficiency of internalisation and recycling. The CTLA-4 YEKM construct 374913-63-0 chemical information remained endocytic albeit with slightly reduced efficiency compared to CTLA-4 YVKM (Figure 6A). However, we also observed impaired recycling by CTLA-4 YEKM (Figure 6B), which was reminiscent of the lower recycling observed for the xenopus CTLA-4 chimera (Figure 6C). Taken together these data suggest that the YVKM motif found in mammalian CTLA-4 25837696 is optimised for both endocytosis and recycling of CTLA-4 and that even relatively subtle variations as found in xenopus CTLA-4 can compromise these 
functions.DiscussionThe role of intracellular trafficking in CTLA-4 function is not well understood, however the high degree of cytoplasmic domain sequence conservation in mammals is intriguing. CTLA-4expressing T cells play an essential role in immune regulation where CTLA-4 is required in order to prevent T cell responses to host tissues. This is achieved by expression of CTLA-4 on specialised regulatory T cells [18]. We have recently shown that a core feature of CTLA-4 biology is its ability to capture ligands from opposing cells, internalise them in a process termed transendocytosis [9]. Such a function presumably involves the specialised intracellular trafficking itinerary. Indeed it is clear that ligands captured by transendocytosis are degraded in the lysosomes in the CTLA-4 expressing cells [9]. Here we have analysed the impact of naturally occurring C-terminal sequence variations of CTLA-4 (found in non-mammals) for their impact on trafficking. We show that C-terminal amino acid sequence variation has an impact on CTLA-4 endocytosis, recycling and degradation. Whilst in general CTLA-4 shows the ability to internalise in all the chimeras tested, we noted that the C-terminus of trout CTLA-4 conferred a substantially reduced efficiency and consequently a much higher plasma membrane expression at steady state. Nonetheless endocytosis could occur through a newly 
identified YGNF motif. Interestingly this YxxF motif is found in several fish species including trout and salmon. Moreover, in zebrafish a YVKF motif is observed which is remarkably similar to the YVKM found in mammalian CTLA-4 [12]. Whilst there are a number of fish genomes undergoing analysis, at present the unequivocal identification of CD28 and CTLA-4 genes is still limited and therefore awaits further characterization. Thus it is plausible that these motifs represent the initial, albeit rather ineffective, emergence of endocytosis in CTLA-4. In contrast to fish, the C.Monstrated no obvious co-localisation with CD63. Collectively these data suggest that degradation of CTLA-4 is related to its endocytic capacity and that the presence of YV/ EKM motif promotes both internalisation and sensitivity to lysosomal degradation. Moreover, trout CTLA-4 contains an alternate tyrosine-based motif that has a reduced efficiency of endocytosis and has a more stable cell surface phenotype when compared to CTLA-4 from human, chicken and xenopus.is seen as an increase in the Alexa647 signal 1676428 over time. Notably, human CTLA-4 and chimeric chicken CTLA-4 (YVKM containing proteins) showed a comparable rate of recycling (Figure 5A and B) whereas chimeric xenopus and trout CTLA-4, which lacked YVKM motif were notably less efficient. Since an acidic residue (E) at the +1 position of YxxM has been suggested to favour an interaction with the lysosomal sorting adaptor AP-3 [17], we mutated the YVKM motif in human CTLA-4 to YEKM and assayed the efficiency of internalisation and recycling. The CTLA-4 YEKM construct remained endocytic albeit with slightly reduced efficiency compared to CTLA-4 YVKM (Figure 6A). However, we also observed impaired recycling by CTLA-4 YEKM (Figure 6B), which was reminiscent of the lower recycling observed for the xenopus CTLA-4 chimera (Figure 6C). Taken together these data suggest that the YVKM motif found in mammalian CTLA-4 25837696 is optimised for both endocytosis and recycling of CTLA-4 and that even relatively subtle variations as found in xenopus CTLA-4 can compromise these functions.DiscussionThe role of intracellular trafficking in CTLA-4 function is not well understood, however the high degree of cytoplasmic domain sequence conservation in mammals is intriguing. CTLA-4expressing T cells play an essential role in immune regulation where CTLA-4 is required in order to prevent T cell responses to host tissues. This is achieved by expression of CTLA-4 on specialised regulatory T cells [18]. We have recently shown that a core feature of CTLA-4 biology is its ability to capture ligands from opposing cells, internalise them in a process termed transendocytosis [9]. Such a function presumably involves the specialised intracellular trafficking itinerary. Indeed it is clear that ligands captured by transendocytosis are degraded in the lysosomes in the CTLA-4 expressing cells [9]. Here we have analysed the impact of naturally occurring C-terminal sequence variations of CTLA-4 (found in non-mammals) for their impact on trafficking. We show that C-terminal amino acid sequence variation has an impact on CTLA-4 endocytosis, recycling and degradation. Whilst in general CTLA-4 shows the ability to internalise in all the chimeras tested, we noted that the C-terminus of trout CTLA-4 conferred a substantially reduced efficiency and consequently a much higher plasma membrane expression at steady state. Nonetheless endocytosis could occur through a newly identified YGNF motif. Interestingly this YxxF motif is found in several fish species including trout and salmon. Moreover, in zebrafish a YVKF motif is observed which is remarkably similar to the YVKM found in mammalian CTLA-4 [12]. Whilst there are a number of fish genomes undergoing analysis, at present the unequivocal identification of CD28 and CTLA-4 genes is still limited and therefore awaits further characterization. Thus it is plausible that these motifs represent the initial, albeit rather ineffective, emergence of endocytosis in CTLA-4. In contrast to fish, the C.
