RESUMO
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.
