Identification of annexin VI as a Ca2+-sensitive CRHSP-28-binding protein in pancreatic acinar cells.
CRHSP-28 is a member of the tumor protein D52 protein family that was recently shown to regulate Ca(2+)-stimulated secretory activity in streptolysin-O-permeabilized acinar cells (Thomas, D. H., Taft, W. B., Kaspar, K. M., and Groblewski, G. E. (2001) J. Biol. Chem. 276, 28866-28872). In the present study, the Ca(2+)-sensitive phospholipid-binding protein annexin VI was purified from rat pancreas as a CRHSP-28-binding protein. The interaction between CRHSP-28 and annexin VI was demonstrated by coimmunoprecipitation and gel-overlay assays and was shown to require low micromolar levels of free Ca(2+), indicating these molecules likely interact under physiological conditions. Immunofluorescence microscopy confirmed a dual localization of CRHSP-28 and annexin VI, which appeared in a punctate pattern in the supranuclear and apical cytoplasm of acini. Stimulation of cells for 5 min with the secretagogue cholecystokinin enhanced the colocalization of CRHSP-28 and annexin VI within regions of acini immediately below the apical plasma membrane. Tissue fractionation revealed that CRHSP-28 is a peripheral membrane protein that is highly enriched in smooth microsomal fractions of pancreas. Further, the content of CRHSP-28 in microsomes was significantly reduced in pancreatic tissue obtained from rats that had been infused with a secretory dose of cholecystokinin for 40 min, demonstrating that secretagogue stimulation transiently alters the association of CRHSP-28 with membranes in cells. Collectively, the Ca(2+)-dependent binding of CRHSP-28 and annexin VI, together with their colocalization in the apical cytoplasm, is consistent with a role for these molecules in acinar cell membrane trafficking events that are essential for digestive enzyme secretion.
Thomas
Diana D H
DD
Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA.
Kaspar
Kala M
KM
Taft
William B
WB
Weng
Ning
N
Rodenkirch
Lance A
LA
Groblewski
Guy E
GE
D000818:Animals; D017317:Annexin A6 / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley
D017317:Annexin A6; D010750:Phosphoproteins; C102584:calcium-regulated heat-stable protein 28