Membrane Fusion Protein Annexin 7: A Common Site of Action for Calcium, Guanosine Triphosphate, Protein Kinase C and Botulinum Toxin Type C in Regulated Exocytosis
Abstract
Numerous studies have shown that regulated exocytosis is activated simultaneously by calcium, guanosine triphosphate (GTP) and protein kinase C (PKC), and that this process is specifically inhibited by botulinum toxins (BoNTs). Although phenomenologically well known, the specific sites of action for these agents in the late stage of exocytosis, membrane fusion, remain unknown. In this research project, we combined both in vitro and in vivo approaches to directly test the effects of these agents on annexin 7. Annexin 7 (ANX7) is a calcium-dependent GTP-activated membrane fusion protein. In a reconstituted membrane fusion system using artificial liposomes, ANX7 membrane fusion activity is substantially increased by the combination of individually optimal concentration of guanine nucleotide and PKC. This increasing ANX7 activity can be distinguished by a simple additive model when comparing activation by either guanine nucleotide or PKC alone. In other in vitro assays, the binding of GTP and its non-hydrolyzable analogues to ANX7 significantly enhance PKC phosphorylation, and conversely PKC phosphorylation markedly potentiates the binding and hydrolysis of GTP by ANX7. While certain other kinases label ANX7 efficiently, they do not substitute for PKC in potentiating GTP binding or membrane fusion. To correlate the in vitro data with exocytotic events in cells, we examined the biochemical profile of endogenous ANX7 in secreting adrenal chromaffin cells. In vivo, both the ratio of ANX7-bound GDP/GTP as well as ANX7 phosphorylation by PKC change in proportion to the extent of catecholamine release from stimulated chromaffin cells. Thus, the stimulatory actions of calcium, GTP and PKC appear to specifically converge on ANX7 to drive membrane fusion activity occurring during exocytosis.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 2002
- Accession Number
- ADA421178
Entities
People
- Hung Caohuy
Organizations
- Uniformed Services University of the Health Sciences