The candidate has developed an everlasting interest in membranes, phosphoinositides (PPI) and phospholipases. The immediate goal is to build a foundation for the research project outlined in this application which will enable, in the long term, to unravel the importance of PPI and phospholipases in cellular functions. Project: Breakdown of PPI by phospholipase C (PLC) in various cells, including platelets, is involved in signal transduction. The HYPOTHESIS is that (a) platelet activating factor (PAF) receptor is coupled via a G-protein to the activation and regulation of the PLC in platelets and (b)exogenous PLC or activation of endogenous PLC cause release of PI- anchored proteins from platelet membrane surfaces. There are four experimental plans. [I] TO DETERMINE MECHANISM(S) OF PAF RECEPTOR-COUPLED ACTIVATION AND DESENSITIZATION OF PLC: (A) to correlate [3H]PAF binding to PLC activation in desensitized platelets as compared to control; (B) to manipulate G-proteins by using NaF, GTPgammas and pertussis toxin and to correlate their influence on PLC activation; (C) to correlate protein kinase C-mediated phosphorylation to the activation and desensitization of PLC. [II] TO DETERMINE MOLECULAR INTERACTIONS AMONG PLC, G-PROTEINS AND PAF RECEPTOR: Platelet membranes will be solubilized and fractionated by column chromatography. [3H]PAF binding, GTPase, 32P-GTP binding and PLC activities will be monitored in fractions to establish their functional associations. [III] TO DETERMINE IMPORTANCE OF PAF RECEPTOR COUPLED ACTIVATION OF PLC IN THE HYPERSENSITIVITY OF DIABETIC HUMAN PLATELETS. [IV] TO DETERMINE THE RELEASE OF PI-ANCHORED MEMBRANE PROTEIN/GLYCOPROTEIN BY EXOGENOUS AND ENDOGENOUS PLC: Release of proteins by PLC (B. thuringiensis) from labelled platelets; identification by SDS-PAGE/fluirography and antibodies; use of differential phase partitioning to monitor PI-anchored proteins after PAF stimulation. The project will provide novel insight(s) in PAF-stimulated PPI turnover and on the importance of PI-anchored proteins in platelets.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Modified Research Career Development Award (K04)
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Physiological Chemistry Study Section (PC)
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University of Missouri-Columbia
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Shukla, S D (1996) Tyrosine kinase activation by PAF leads to downstream gene expression. Adv Exp Med Biol 416:153-5
James-Kracke, M R; Sexe, R B; Shukla, S D (1994) Picomolar platelet-activating factor mobilizes Ca to change platelet shape without activating phospholipase C or protein kinase C;simultaneous fluorometric measurement of intracellular free Ca concentration and aggregation. J Pharmacol Exp Ther 271:824-31
Jones, A W; Shukla, S D; Geisbuhler, B B (1993) Stimulation of phospholipase D activity and phosphatidic acid production by norepinephrine in rat aorta. Am J Physiol 264:C609-16
Zhu, C Y; Shukla, S D (1993) Increased tyrosine kinase activity in pp60c-src immunoprecipitate from platelet activating factor stimulated human platelets: in vitro phosphorylation of a synthetic peptide. Life Sci 53:175-83
Dhar, A; Shukla, S D (1993) Tyrosine kinases in platelet signalling. Br J Haematol 84:1-7
Thurston Jr, A W; Rhee, S G; Shukla, S D (1993) Role of guanine nucleotide-binding protein and tyrosine kinase in platelet-activating factor activation of phospholipase C in A431 cells: proposal for dual mechanisms. J Pharmacol Exp Ther 266:1106-12
Shukla, S D; Paul, A; Klachko, D M (1992) Hypersensitivity of diabetic human platelets to platelet activating factor. Thromb Res 66:239-46
Shukla, S D (1992) Platelet-activating factor receptor and signal transduction mechanisms. FASEB J 6:2296-301
Tripathi, Y B; Lim, R W; Fernandez-Gallardo, S et al. (1992) Involvement of tyrosine kinase and protein kinase C in platelet-activating-factor-induced c-fos gene expression in A-431 cells. Biochem J 286 ( Pt 2):527-33
Dhar, A; Shukla, S D (1991) Involvement of pp60c-src in platelet-activating factor-stimulated platelets. Evidence for translocation from cytosol to membrane. J Biol Chem 266:18797-801

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