This application for a FIRST AWARD is based on the hypothesis that the motility of polymorphonuclear leukocyte (PMN) on the vitronectin (Vn) substrate is regulated by the changes in intracellular calcium and calmodulin-dependent phosphatase, Calcineurin, via a Vn (vitronectin) receptor, that is, alphavbeta3, the alphav being an isoform unique to PMNs. It further proposes that in addition to the beta3, beta2 integrin receptor is involved in the phosphorylation- and calcium-dependent intracellular signalling pathways, and both these receptors are critical to the attachment/detachment processes related to PMN motility on vitronectin substrate. The studies are proposed under three specific aims. In the first specfic aim, characterization of alphav of PMN will be carried out with the anticipation that it is unique to PMN, and it differs in its N-terminal segment from alphav subunits distributed in other tissues. The characterization will include first generating the cDNA, from isolated PMN s RNA, and then reverse transcribing them by the polymerase chain reaction (PCR). After isolation of the alphav cDNA, it will be cleaved into short fragments by restriction endonucleases for sequence analyses. In addition to the nucleotide sequencing, peptide sequencing of the N-terminus will be performed. The second specific aim relates to the elucidation of the role of beta2 integrin in PMN attachment with the background contention that beta2 receptor is required for beta3-mediated attachment, and both beta2 and beta3 are essential to PMN attachment on the vitronectin substrate. In these experiments, the cell and cell lines deficient in beta2 subunit (leukocyte adhesion deficiency patients) and beta3 subunit (HL-60 cell line) receptors will be utilized. Various transfection experiments are proposed in this section to ascertain whether the beta3-deficient cells revert to their normal behavior after transfection with beta3 cDNA. Also, by antibody (AP5) manipulation it will be determined that if PMNs from LAD patients with beta2 receptor deficiency can be forced to attach to the Vn substrate utilizing beta3 receptor. In the third specific aim the role of phosphokinase-C (PKC) and calcineurin (a phosphatase) on the beta2 phosphorylation and beta3-mediated PMN motility will be examined.
This specific aim i s based on the contention that the motility of the PMN is related to the balance between the phosphorylation by PKC and dephosphorylation or phosphatase activity of calcineurin. The experiments with specific inhibitors of PKC and calcineurin will be performed to ascertain the phosphorylation of beta2. They will be followed by experiments in which signalling events distal to the beta2 phosphorylation will assessed, and these include phosphorylation of paxillin and changes in the intracellular calcium. Finally, relationship between beta2 phosphorylation and beta3-mediated attachment or detachment will be assessed.
