CHARACTERIZATION OF THE LEUKOCYTE INS (1,4,5)P3 RECEPTOR

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Studies will deal with a working model for mobilization of intracellular calcium by inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) in chemoattractant stimulated neutrophils. A specific receptor for Ins-1,4,5-P3 in saponin-permeabilized rabbit neutrophils and on microsomes has been identified. The characteristics of this receptor suggest that it is the physiological receptor in neutrophils which regulates the release of sequestered calcium from the endoplasmic reticulum. In an attempt to unify the data into a testable hypothesis, a model of an Ins-1,4,5-P3 dependent, GTP-regulated Ca2+ channel in the endoplasmic reticulum membrane is proposed. The major emphasis of the planned studies is placed on the purification and characterization of a GTP- regulated, Ins-1,4,5-P3 receptor from cell homogenates and microsomal fractions from DMSO-differentiated human promyelocytic HL-60 cells. Solubilized receptors will be purified by conventional ion exchange and molecular sieve chromatography using as an assay for receptor the specific binding of 32P-Ins- 1,4,5-P3 to isolated fractions following reconstitution into lipid vesicles and detergent removal. Attempts will be made to synthesize an affinity support using p-amino benzoiacid derivatized inositol 1,4,5-trisphosphate coupled to an activated CH-sepharose 4B matrix. Using the reconstituted, purified receptor, the mechanism of action of Ins-1,4,5-P3 in stimulating Ca2+ release and the modulatory role of GTP will be probed. The Ins-1,4,5-P3 binding data will be analyzed to obtain equilibrium (Kd and receptor number) and kinetic (thermodynamic) constants. These will be compared to those found for permeable cells. The purified receptor will be used to raise specific antibodies to be used in functional assays and purification protocols. Using oligonucleotide probes synthesized from N-terminus sequence data derived from trypsin fragments of the receptor, cDNA clones will be selected. Sequence data will then be derived. These studies may ultimately enable a better understanding of intracellular regulation of Ca2+ release in neutrophils.