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J. Biol. Chem., Vol. 276, Issue 31, 28866-28872, August 3, 2001
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From the Department of Nutritional Sciences, University of
Wisconsin, Madison Wisconsin 53706
CRHSP-28 is a
Ca2+-regulated heat-stable phosphoprotein, abundant
in the apical cytoplasm of epithelial cells that are specialized in
exocrine protein secretion. To define a functional role for the protein
in pancreatic secretion, recombinant CRHSP-28 (rCRHSP-28) was
introduced into streptolysin-O-permeabilized acinar cells, and amylase
secretion in response to elevated Ca2+ was determined.
Secretion was enhanced markedly by rCRHSP-28 over a time course that
closely corresponded with the loss of the native protein from the
intracellular compartment. No effects of rCRHSP-28 were detected until
~50% of the native protein was lost from the cytosol. Secretion was
enhanced by rCRHSP-28 over a physiological range of Ca2+
concentrations with 2-3-fold increases in amylase release occurring in
response to low micromolar levels of free Ca2+. Further,
rCRHSP-28 augmented secretion in a concentration-dependent manner with minimal and maximal effects occurring at 1 and 25 µg/ml, respectively. Covalent cross-linking experiments demonstrated that native CRHSP-28 was present in a 60-kDa complex in cytosolic fractions and in a high molecular mass complex in particulate fractions, consistent with the slow leak rate of the protein from streptolysin-O-permeabilized cells. Probing acinar lysates with rCRHSP-28 in a gel-overlay assay identified two CRHSP-28-binding proteins of 35 (pp35) and 70 kDa (pp70). Interestingly, preparation of
lysates in the presence of 1 mM Ca2+ resulted
in a marked redistribution of both proteins from a cytosolic to a
Triton X-100-insoluble fraction, suggesting a
Ca2+-sensitive interaction of these proteins with the
acinar cell cytoskeleton. In agreement with our previous study
immunohistochemically localizing CRHSP-28 around secretory granules in
acinar cells, gel-overlay analysis revealed pp70 copurified with acinar
cell secretory granule membranes. These findings demonstrate an
important cell physiological function for CRHSP-28 in the
Ca2+-regulated secretory pathway of acinar cells.
CRHSP-28 Regulates Ca2+-stimulated Secretion in
Permeabilized Acinar Cells*
*
This work was supported by Grants WISO4221 and WIS04444 from
the United States Department of Agriculture Cooperative State Research
Education and Extension Service Program, American Cancer Society
Institutional Research Grant IRG-58-011-42-2 and National Science
Foundation Grant MCB-0094154 (to G. E. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Nutritional Sciences, University of Wisconsin, 1415 Linden Dr.,
Madison, WI 53706. Tel.: 608-262-0884; Fax: 608-262-5830;
E-mail: groby@nutrisci.wisc.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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