CRHSP-28 regulates Ca2+-stimulated secretion in permeabilized acinar cells

CRHSP-28 is a Ca2+-regulated heat-stable phosphoprotein, abundant in the ap ical cytoplasm of epithelial cells that are specialized in exocrine protein secretion. To define a functional role for the protein in pancreatic secre tion, recombinant CRHSP-28 (rCRHSP-28) was introduced into streptolysin-O-p ermeabilized 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 similar to 50% of the native protein was lost from the cytosol. Secretion w as enhanced by rCRHSP-28 over a physiological range of Ca2+ concentrations with 2-3-fold increases in amylase release occurring in response to low mic romolar levels of free Ca2+. Further, rCRHSP-28 augmented secretion in a co ncentration-dependent manner with minimal and maximal effects occurring at 1 and 25 mug/ml, respectively. Covalent cross-linking experiments demonstra ted that native CRHSP-28 was present in a 60-kDa complex in cytosolic fract ions and in a high molecular mass complex in particulate fractions, consist ent with the slow leak rate of the protein from streptolysin-O-permeabilize d cells. Probing acinar lysates with rCRHSP-28 in a gel-overlay assay ident ified two CRHSP-28-binding proteins of 35 (pp35) and 70 kDa (pp70). Interes tingly, preparation of lysates in the presence of 1 mm Ca2+ resulted in a m arked redistribution of both proteins from a cytosolic to a Triton X-100-in soluble fraction, suggesting a Ca2+- sensitive interaction of these protein s with the acinar cell cytoskeleton. In agreement with our previous study i mmunohistochemically localizing CRHSP-28 around secretory granules in acina r cells, gel-overlay analysis revealed pp70 copurified with acinar cell sec retory granule membranes. These findings demonstrate an important cell phys iological function for CRHSP-28 in the Ca2+-regulated secretory pathway of acinar cells.