Delineation of the Oligomerization, AP-2 Binding, and Synprint Binding Region of the C2B Domain of Synaptotagmin

• Published in: The Journal of biological chemistry 1998-12, Vol.273 (49), p.32966-32972
• Main Authors: Chapman, Edwin R., Desai, Radhika C., Davis, Anson F., Tornehl, Christopher K.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Biopolymers; Calcium-Binding Proteins; DNA-Binding Proteins - metabolism; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Models, Molecular; Molecular Sequence Data; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - metabolism; Protein Binding; Rats; Sequence Homology, Amino Acid; Synaptotagmin I; Synaptotagmins; Transcription Factor AP-2; Transcription Factors - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.49.32966

Biochemical and genetic studies indicate that synaptotagmin I functions as a Ca2+ sensor during synaptic vesicle exocytosis and as a membrane receptor for the clathrin adaptor complex, AP-2, during endocytosis. These functions involve the interaction of two conserved domains, C2A and C2B, with effector proteins. The C2B domain mediates Ca2+-triggered synaptotagmin oligomerization, binds AP-2 and is important for the interaction of synaptotagmin with Ca2+ channels. Here, we report that these are conserved biochemical properties: Ca2+ promoted the hetero-oligomerization of synaptotagmin I with synaptotagmins III and IV, and all three synaptotagmin isoforms bound the synprint region of the α1B subunit of N-type Ca2+ channels. Using chimeric and truncated C2 domains, we defined a common region of C2B that mediates oligomerization and AP-2 binding. Within this region, two adjacent lysine residues were identified that were critical for synaptotagmin oligomerization, AP-2, and synprint binding. Competition experiments demonstrated that the synprint fragment was an effective inhibitor of synaptotagmin oligomerization and also blocked binding of synaptotagmin to AP-2. In a model for the structure of C2B, the common effector binding site localized to a putative Ca2+-binding loop and a concave region formed by two β-strands. These studies provide the first structural information regarding C2B target protein recognition and provide the means to selectively disrupt synaptotagmin-effector interactions for functional studies.