The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis

Abstract

One characteristic linking members of the synaptotagmin family to endocytosis is their ability to bind the heterotetrameric AP2 complex via their C2B domain. By using CD4/synaptotagmin 1 chimeras, we found that the internalization signal of synaptotagmin 1 lies at the extreme COOH-terminus of the protein and can function in the absence of the C2B domain that contains the AP2 binding site. However, although not essential for internalization, the C2B domain of synaptotagmin 1 appeared to control the recognition of the internalization motif. By mutagenesis, two sites have been identified that modify regulation by the C2B domain in the neuroendocrine PC12 cell line. Mutation of a dilysine motif in the β sandwich core of the domain eliminates endocytosis. This site is known to be a site of protein–protein interaction. Mutations in the calcium binding region, or in its close proximity, also affect internalization in PC12 cells. In fibroblasts, the C2B domain inhibits the COOH-terminal internalization signal, resulting in an absence of internalization in those cells. Thus, internalization of synaptotagmin 1 is controlled 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.

Effects of synaptotagmin reveal two distinct mechanisms of agonist‐stimulated internalization of the M4 muscarinic acetylcholine receptor

“…As shown in Figure 1d-g, the distribution of the two signals was identical, and both wild-type and mutant synaptotagmin were expressed almost exclusively at the plasma membrane. This intracellular sorting contrasts sharply with that normally seen in neurons and neuroendocrine cells, where synaptotagmin is delivered to synaptic vesicles and secretory granules, respectively (Matthew et al, 1981), but is in accord with the observed targeting of synaptotagmin to the plasma membrane in Chinese hamster ovary (CHO) cells (Jarousse & Kelly, 2001). We observed similar targeting using wild-type…”

O-glycosylation is essential for intracellular targeting of synaptotagmins I and II in non-neuronal specialized secretory cells

“…Therefore, internalization from the plasma membrane might serve a crucial step in defining the final localization of these homologues. Indeed, both internalization signals and inhibitory signals co-embedded within synaptotagmin C2 domains as well as N-glycosylation of the luminal domain have been proposed to govern synaptotagmin internalization (Han et al, 2004;Dasgupta and Kelly, 2001;Jarousse and Kelly, 2001). Alternatively, in non-neuronal secretory cells, synaptotagmins might be delivered to their secretory granule destination directly from the trans-Golgi network (TGN) bypassing the plasma membrane and the need for internalization.…”

Internalization signals in synaptotagmin VII utilizing two independent pathways are masked by intramolecular inhibitions

“…For the CD4-synaptotagmin constructs, a CD4 fragment (corresponding to residues 1-426) encoding the lumenal, transmembrane and 12 amino acids of the cytoplasmic region of the human CD4 was amplified by PCR from pBMN-Syt 1 (Jarousse and Kelly, 2001a). The primers were chosen so that the CD4 coding region was downstream of a BamHI restriction site and upstream of a BstBI restriction site followed by a stop codon and SalI restriction site.…”

“…Surprisingly, the internalization signal of synaptotagmin I is not identical to and does not require the AP-2-binding site. The internalization signal was found to be in a region of the C2B domain near the C-terminus (Blagoveshchenskaya et al, 1999;Jarousse and Kelly, 2001a). The region of the C-terminus critical for endocytosis was the WHXL motif (N. Jarousse, J. Wilson, D. Arac, J. Rizo and R.B.K., unpublished).…”

“…The region of the C-terminus critical for endocytosis was the WHXL motif (N. Jarousse, J. Wilson, D. Arac, J. Rizo and R.B.K., unpublished). Regulation of this internalization signal was shown to be responsible for tissuespecific endocytosis of synaptotagmin I (Jarousse and Kelly, 2001a). Here we show that synaptotagmin VII is not actively internalized in neuronal, fibroblast and epithelial cell types despite having the AP-2-binding site and the WHXL motif, which are important for internalization of synaptotagmin I.…”