Abstract
CRHSP-28 is a Ca(2+)-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 Ca(2+) 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 approximately 50% of the native protein was lost from the cytosol. Secretion was enhanced by rCRHSP-28 over a physiological range of Ca(2+) concentrations with 2-3-fold increases in amylase release occurring in response to low micromolar levels of free Ca(2+). Further, rCRHSP-28 augmented secretion in a concentration-dependent manner with minimal and maximal effects occurring at 1 and 25 microg/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 Ca(2+) resulted in a marked redistribution of both proteins from a cytosolic to a Triton X-100-insoluble fraction, suggesting a Ca(2+)-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 Ca(2+)-regulated secretory pathway of acinar cells.
Highlights
Introduction of rCRHSP28 into permeabilized acini had no effect on secretion during the first 10 min of Ca2ϩ-stimulation; rCRHSP-28 augmented secretion by greater than 160% of untreated cells when measured at 20 and 30 min
The addition of rCRHSP-28 to cells that had been pre-permeabilized for 10 min, washed, and resuspended in incubation buffer containing the protein had no significant effect on amylase secretion
Introduction of rCRHSP-28 into SLO-permeabilized acini markedly enhanced Ca2ϩ-stimulated amylase release with a time course that corresponded to the loss of the native protein from the intracellular compartment
Summary
Introduction of rCRHSP28 into permeabilized acini had no effect on secretion during the first 10 min of Ca2ϩ-stimulation (filled circles); rCRHSP-28 augmented secretion by greater than 160% of untreated cells when measured at 20 and 30 min. It is proposed that CRHSP-28 functions in acinar cell secretion by modulating the delivery of secretory granules to the apical plasma membrane via dynamic interactions with the pp70 protein.
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