The stimulatory effect of calpactin (annexin II) on calcium-dependent exocytosis in chromaffin cells: requirement for both the N-terminal and core domains of p36 and ATP.
Calpactin, or calpactin heavy chain (p36), reconstitute secretion in digitonin-permeabilized adrenal chromaffin cells after a reduction in their secretory potential resulting from the loss of cytosolic components. We have characterized the stimulatory effect of p36, which resulted in an increase in both the extent and the rate of exocytosis. A mixture of other annexins (p70 and p32) did not have any effect on secretion at similar or greater concentrations than p36. Controlled proteolysis of p36 using chymotrypsin was carried out, and the 33,000 molecular weight core and 3000 molecular weight tail peptide isolated. In contrast to p36, p33 had no effect on exocytosis, even at high calcium concentrations. The N-terminal tail peptide and a synthetic peptide based on the tail of p36 [Ac-calpactin-(1-15)-NH2] had no effect on endogenous secretion, or secretion stimulated by exogenous p36. The results show that both the tail and core domains are required for p36 to stimulate exocytosis. The tail domain is unlikely to be required for interaction with cellular components but probably has a regulatory effect on the core domain. Endogenous secretion and the stimulatory effect of p36 were markedly inhibited by depletion of ATP. The ATP requirement for p36 action was not due to a requirement for phosphorylation by protein kinase C (PKC), since the PKC inhibitor staurosporine partially inhibited endogenous secretion but did not affect the stimulation of exocytosis due to exogenous p36.
Ali
S M
SM
Physiological Laboratory, University of Liverpool, U.K.
Burgoyne
R D
RD
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000313:Adrenal Medulla / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D017302:Annexins; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D002478:Cells, Cultured; D005089:Exocytosis / Q000187:drug effects*; D008565:Membrane Proteins / Q000378:metabolism / Q000494:pharmacology*; D008954:Models, Biological; D011493:Protein Kinase C / Q000037:antagonists & inhibitors
D017302:Annexins; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D002118:Calcium