This application proposes to address a key problem in Ca+2 signaling?the identification of the intracellular nicotinic acid adenine dinucleotide phosphate (NAADP) receptor. NAADP signaling elicits cytosolic Ca2+ mobilization in sea urchin eggs, in primary mouse T cells, and in a wide variety of human tissues. NAADP is the most potent Ca+2 mobilizing intracellular 2nd messenger, and human as well as invertebrate cells release Ca2+ from lysosome like acidic vesicles in response to nanomolar concentrations of NAADP, arguing for the importance and generality of this signaling mechanism. NAADP initiated Ca+2 sparks may trigger further Ca+2 induced Ca2+ release from the endoplasmic reticulum through other ion-channels responsive to inositol triphosphate (IP3) and cyclic ADP-ribose. Our objective will be to identify NAADP binding proteins in sea urchin and in vertebrates. The results of our previous photoaffinity labeling studies identified specific high affinity NAADP binding sites on 45, 40, and 30 kDa proteins in sea urchin egg homogenates and on 23/24 kDa proteins in cultured human cells. We propose to purify NAADP binding proteins from solubilized sea urchin egg homogenates through the application of affinity chromatography combined with standard biochemical separations. In parallel, we will apply a direct affinity-based separation on cytosolic Jurkat cell NAADP binding proteins. Our objective is to isolate 0.1-1 g quantities of each purified NAADP binding protein and then to identify each by obtaining partial sequences using mass spectrometry. Alternative approaches to the purification and isolation of NAADP binding proteins are available through the use of photoactive NAADP-biotin conjugates, which by forming a covalent bond to the binding protein could be affinity purified using more aggressive conditions. Photoactive and clickable-NAADP analogs are available as a second alternative covalent labeling strategy if the photoactive NAADP-biotin conjugates are not recognized by the binding proteins at low concentration. We envision that NAADP binding proteins control Ca2+ ion-channels (suggested by some to be the two-pore channel) through protein-protein interactions. The identification of these proteins is now a high priority. Once NAADP binding proteins are identified, their function will be validated by expression and characterization of the expressed proteins. Additional experiments are proposed to determine the role of NAADP binding proteins in NAADP mediated Ca2+ release in our model systems. These will involve binding protein knockdown using siRNAs, characterization of the effects of overexpression of recombinant proteins in cultured cells, and cellular localization.
Changes in the intracellular calcium-ion concentration are a fundamental cell regulatory mechanism. Nicotinic acid adenine dinucleotide 2?-phosphate (NAADP) is one of several interacting intracellular 2nd messengers that mobilize calcium-ion to control biological responses. Our goal is to use a chemical and biochemical approach to identify the receptor for NAADP and apply this discovery to better understand this fundamental cell regulatory mechanism.
