CRHSP-28 and Exocrine Secretion in the Digestive System

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

CRHSP-28 is a novel Ca 2+ -sensitive regulatory protein, abundant in epithelial cells that are specialized in exocrine protein secretion. This includes Paneth cells that secrete antimicrobial peptides, chief and pancreatic cells that secrete digestive enzymes, lacrimal cells for corneal function and goblet cells that secrete mucin for tissue barrier protection. Introduction of recombinant CRHSP-28 into permeabilized acinar cells completely reverses the decreased Ca 2+ -dependent secretory capacity caused by the leakage of cytosolic proteins from the cell interior, indicating a direct role for CRHSP-28 in the secretory pathway. Consistent with a coiled coil interaction motif near its amino terminus, CRHSP-28 exists as a dimer in cytosolic extracts, and is associated with a large molecular complex in membrane/cytoskeleton fractions. Further, CRHSP-28 is concentrated around secretory granules in the apical regions of exocrine cells, and specifically associates with a 70 kDa binding protein (pp70) that co-purifies with secretory granule membranes. Significantly, soluble forms of this binding protein associate with taxol-stabilized microtubules when analyzed in the absence of Ca 2+ ; however, when exposed to elevated Ca 2+ , pp70 undergoes a complete translocation to an actin-rich detergent insoluble fraction. Based on biochemical and immunohistochemical data, CRHSP-28 and its associated proteins are proposed to regulate the delivery and movement of secretory granules through the subapical actin cytoskeleton in a Ca 2+ /calmodulin dependent manner. The triggering mechanism for this effect is theorized to entail the acute serine phosphorylation of CRHSP-28 by the multifunctional CaM kinase II. Thus, the primary objective of this project is to elucidate the precise biochemical and molecular mechanisms by which this novel CRHSP-28-mediated pathway regulates Ca 2+ -stimulated exocrine secretion. The Specific Aims of this project are: 1). To test the idea that the Ca2+/calmodulin regulated phosphorylation of CRHSP-28 is a key signaling mechanism by which the protein modulates secretion. Experiments will first address the identity of the Ca 2+ -sensitive serine phosphorylation sites on CRHSP-28. Phosphorylation sites will be mapped in vitro and in intact cells, and verified by transfection of phosphorylation mutants into cultured T84 cells. The effects of normal and mutated forms of CRHSP-28 will then be evaluated for their ability to alter secretagogue-stimulated mucin release from these cells. Similarly, CRHSP-28 mutants will be expressed and purified in bacteria and tested in permeabilized acinar cells to assess their effects on digestive enzyme secretion. 2). To evaluate the concept that CRHSP-28 functions in the secretory pathway of exocrine cells through dynamic interactions with other key regulatory proteins. These studies will be centered on identifying the molecular identity of the 70 kDa CRHSP-28 binding protein that copurifies with secretory granule membranes, binds to taxol-stabilized microtubules, and translocates to detergent-insoluble cytoskeletal fractions in the presence of elevated Ca 2+ . CRHSP-28 affinity chromatography and cDNA library screening will be used as purification techniques. Following cloning, tagged proteins will be expressed in cells to characterize the molecular interactions of CRHSP-28 with itself (as a dimer) and the 70 kDa binding protein using coimmunoprecipitations and dual immunofluorescence labeling. This approach will allow for identification of the specific sites that mediate these interactions, as well as the effects of second messengers and phosphorylation in regulating binding. Complementary studies will be conducted using tagged recombinant proteins for in vitro binding assays. Identification and characterization of CRHSP-28 protein interactions within the cell is key to understanding its regulatory function.