However, in preliminary experiments, cotransfection of CHO cells with synaptotagmin and AP180 or dynamin 1 has not activated the internalization of synaptotagmin (data not shown). by the presence of a latent internalization signal in the COOH-terminal region and a regulatory region in the C2B domain. We propose that internalization of synaptotagmin 1 is regulated in this way to allow it to couple the processes of endocytosis and calcium-mediated exocytosis in cells of the neuroendocrine lineage. We selected several clones with relatively low expression levels. The total levels of expression were measured by flow cytometry after the staining of permeabilized cells with 604.1 antibody followed by a fluorescent secondary antibody. We examined these same clones in the internalization assay described above. Regardless of the total level of expression, the amount of 604.1 remaining at the surface after 10 min at 37C was between 75 and 100% of the initial value, even in a clone whose expression level matched that of PC12 (Fig. 1 c). That was in clear contrast with the Spinosin result obtained in PC12 cells where 40% was detected at the surface after 10 min at 37C. Our results were confirmed using a conventional endocytosis assay based on internalization of 125I-604.1 into an acid-resistant pool (Fig. 1 d). Synaptotagmin 1 was not internalized when transfected into CHO cells, or into human embryonic kidney (HEK)* cells, another nonneuronal cell line. It thus appears as if nonneuronal cells lack some components or pathways involved in synaptotagmin 1 internalization. Open in a separate window Figure 1. Comparison of synaptotagmin 1 internalization in CHO and PC12 cells. (a) wtPC12 or (b) CHO stably transfected with synaptotagmin 1 (CHOsyn1) were labeled at 4C with the 604.1 antibody and then moved to 37C for the Spinosin indicated periods. Cells were cooled to 4C and antibody remaining at the surface after the 37C incubation was detected with a fluorescein-conjugated secondary antibody. The intensity of fluorescence was determined by flow cytometry. Data were expressed as the percentage of the initial value at = 0. (c) The expression level of synaptotagmin 1 in different CHOsyn1 clones was determined by flow cytometry after permeabilization of the cells and staining with 604-1 antibody. These values are expressed along the x-axis. The same clones were then analyzed for internalization of synaptotagmin 1 using the same assay as in panels a and b. The values obtained after 10 min at 37C correspond to the y-axis. The same measurements were done in parallel on PC12 cells. (d) wt PC12, CHOsyn1, and HEK cells stably expressing synaptotagmin 1 (HEKsyn1) were examined for internalization of synaptotagmin 1 using 125I -604.1 antibody. Cells were labeled at 4C and shifted to Spinosin 37C for different Rabbit polyclonal to TSP1 time points. The internalized antibody was determined by surface acid stripping and expressed as a fraction of total cell associated counts. Each time point was done in triplicate. In this and subsequent figures, when standard deviations are not apparent, they were too small to be represented graphically. Internalization of synaptotagmin 1 in PC12 cells is mediated by an internalization signal present in the COOH-terminal Spinosin domain The lack of internalization of synaptotagmin 1 in CHO cells suggested that synaptotagmin 1 might be internalized by a neuron-specific sorting motif recognized by components present only in neurons. Since the C2B domain is known to bind AP2 it might contain the internalization signal. The AP2 binding site in the C2B domain has been mapped in a region between residues 296 and 328 (Chapman et al., 1998). To identify the cytoplasmic domain responsible for internalization of synaptotagmin 1 in PC12 cells, we generated constructs containing the lumenal and transmembrane domain of the CD4 molecule fused to different cytoplasmic regions of synaptotagmin 1 (Fig. 2) . Each construct was stably expressed in PC12 cells by retroviral infection and tested for internalization using uptake of 125I -Q4120, Spinosin a well characterized antibody directed against the external part of CD4. Cell surface CD4 was labeled at 4C and the antibody was allowed to internalize at 37C for 10 min. Internalization of 125I -Q4120 was assessed as acid-resistant bound antibody. As expected from previous studies (Pelchen-Matthews et al., 1991), a CD4 tailess construct was only poorly endocytosed (Fig. 3 a). Its internalization was efficiently promoted by fusion to the cytoplasmic domain of synaptotagmin 1 containing the two C2 domains and the COOH-terminal region (CD4-C2AB-CT). Open in a separate window Figure 2. CD4Csynaptotagmin 1 constructs. A CD4 tailess fragment (human CD4 residues 1C426) was fused to different domains of.