Cellular stimuli (e.g. hormones, transmitters, mitogens, chemotactic factors and other agonists) whose actions involve the mobilization of intracellular Ca2+ as a mediator of cellular responses stimulate the metabolism of membrane phosphoinositides. As a result, the membrane lipid phosphatidylinositol 4,5-bisphosphate is hydrolyzed to generate the product inositol 1,4,5-trisphosphate (IP3). IP3 has been proposed to be the second messenger for the release of intracellular stores of Ca2+. IP3 has been found to release Ca2+ in several types of permeabilized cells, and from isolated membrane fractions from a few cell types. Microinjection of IP3 into egg cells also releases internal Ca2+. The molecular site of action of IP3 is unknown, and the physiological role of IP3 in stimulus-response coupling is yet to be fully established. The available evidence indicates the existence of specific membrane IP3-binding sites and an IP3- activated Ca2+ channel. Our approach to this problem is to employ the powerful hybridoma technology for the production of monoclonal antibodies to identify and isolate the IP3- receptor/channel and to further investigate the physiological role of IP3 in cellular physiology. Our approach is to immunize mice with a purified platelet membrane fraction that releases Ca2+ in response to IP3, and thereby to obtain polyclonal and then monoclonal antibodies that effect the function of the IP3-receptor/channel. Aided by assays (ELISA) that detect antibodies to platelet membrane antigens, and a bioassay that reliably measures Ca2+ release by IP3, we have succeeded in cloning several hybridomas which produce monoclonal antibodies that can completely block the action of IP3. In this study our objectives are to produce and purify a variety of monoclonal antibodies that block the Ca2+-mobilizing action IP3, and to employ these antibodies to: (1) identify the molecular site of action of IP3 (i.e. the receptor/channel), (2) to isolate, purify and characterize the IP3-receptor/channel, of IP3- receptor/channel, and (3) to test the physiological significance of IP3 by studying responses to stimulation in intact cells (e.g. eggs, photoreceptor cells, etc.) that have been microinjected with monoclonal antibodies.
| O'Rourke, F; Feinstein, M B (1990) The inositol 1,4,5-trisphosphate receptor binding sites of platelet membranes. pH-dependency, inhibition by polymeric sulphates, and the possible presence of arginine at the binding site. Biochem J 267:297-302 |
| O'Rourke, F; Zavoico, G B; Feinstein, M B (1989) Release of Ca2+ by inositol 1,4,5-trisphosphate in platelet membrane vesicles is not dependent on cyclic AMP-dependent protein kinase. Biochem J 257:715-21 |
| O'Rourke, F; Zavoico, G B; Smith Jr, L H et al. (1987) Stimulus-response coupling in a cell-free platelet membrane system. GTP-dependent release of Ca2+ by thrombin, and inhibition by pertussis toxin and a monoclonal antibody that blocks calcium release by IP3. FEBS Lett 214:176-80 |
