A SURVEY OF GTP-BINDING PROTEINS AND OTHER POTENTIAL KEY REGULATORS OF EXOCYTOTIC SECRETION IN EOSINOPHILS - APPARENT ABSENCE OF RAB3 AND VESICLE FUSION PROTEIN HOMOLOGS

We set out to identify potential key regulators of exocytotic fusion i n the eosinophil, in the knowledge that granule exocytosis can be stim ulated in these cells by intracellular application of nonhydrolyzable analogues of guanosine triphosphate, with Ca2+ acting as a modulator o f guanine nucleotide-dependent secretion. To screen for GTP-binding pr oteins, guinea pig eosinophils were purified from peritoneal washings and subjected to western blotting analysis using specific immune sera raised against recombinant proteins or consensus peptide sequences wit hin proteins of interest. We found a number of heterotrimeric G protei ns (G alpha(i3), G alpha(o), G alpha(q11) G alpha(s) and G beta subuni ts) and members of the small GTP-binding proteins expressed in eosinop hils, Two subtypes of G-protein alpha subunits (G alpha(i1) and G alph a(z)) could not be detected. Separation of subcellular organelles from homogenized eosinophils by density gradient centrifugation revealed t hat all of the detected GTP-binding proteins were mainly expressed in fractions containing peak plasma membrane and Golgi marker enzyme acti vities, while GP subunits were also detected in secretory granule frac tions. However, isoforms of Rab3, a putative GTP-binding regulator of exocytotic fusion, were undetectable in eosinophils. Neither, with the exception of syntaxin-3, could we detect any of the proteins belongin g to the proposed synaptic vesicle fusion complex (SNAP-25; synaptobre vin (VAMP) and its non-neuronal homologue, cellubrevin; synaptophysin; synaptotagmin). The results from this study, based on western blottin g, suggest that eosinophils express a different class of exocytotic fu sion complex proteins from those found in neuronal tissues, although a number of potential candidates fulfilling the role of GE were identif ied in this important inflammatory cell.