This is a request for five years funding of a competitive renewal to continue studies of the mechanisms used by P- selectin to sort into secretory storage granules and to be endocytosed from the cell surface.P- selectin is a transmembrane glycoprotein which was independently discovered by Dr. McEver and also by Bruce and Barbara Furie. It is located in alpha granules of platelets and Weibel-Palade bodies of endothelial cells. Dr. McEver has found that in response to endothelial agonists such as thrombin, P- selectin rapidly redistributes to the cell surface where it serves asan adhesion receptor for leukocytes.Dr. McEver has also found that following expression, the protein is endocytosed from the plasma membranes of the activated cells. The application is to study this trafficking of P-selectin. Two observations made during the course of his studies has prompted Dr. McEver to go in this direction. The first is that P- selectin, transfected into heterologous cells, is capable of sorting into secretory organelles, much like it is when it is expressed in endothelial cells. Further studies showed that the necessary and sufficient information for this sorting property was contained in the short, 35-aa cytoplasmic tail of P-selectin. Most convincingly, this tail, linked to TF, was found to also direct this molecule into the storage organelles of the transfected cells which - normally contain secretory organelles. Dr. McEver claims that this is the first example of a protein that contains a positive sorting signal in the regulated secretory pathway. The second series of observations on trafficking relates to the endocytic property of P- selectin in endothelial cells. When P-selectin was stably transfected into CHO, it too was endocytosed. Deletion of the cytoplasmic domain caused a marked decrease in endocytosis. These data indicate that the cytoplasmic domain of P-selectin contains dual, possibly independent signals of P-selectin trafficking: one which sorts the protein into secretory organelles during biosynthesis; the other to recover the protein from the cell surface following secretion. The present proposal is designed to identify the structural features of the protein that are responsible for these two activities.
The First Aim i s to characterize the itinerary of newly synthesized P- seIectin in cells containing the regulated secretory pathway. The itinerary of P-selectin will be measured by pulse-chase studies in conjunction with subcellular fractionation,to measure the kinetics of P- selectin distribution to granules, and cell-surface biotinylation, to measure the kinetics of P-selectin distribution to the plasma membrane. Preliminary data suggests further that in some cells there is a degradation of P-selectin as well and if this holds up, Dr. McEver will also study the kinetics of P-selectin targeting to lysozomes.
The Second Aim i s to compare the signals that sort P-selectin into secretory granules with those required for endocytosis. The sorting and endocytosis signals will be examined by site-directed mutagenesis, following the trafficking patterns of the expressed proteins.The structures of wild-type and mutant cytoplasmic domain peptides will be examined by circulardichroism and intrinsic tyrosine fluorescence in an attempt to identify structural features of the cytoplasmic domain which are responsible for the two trafficking routes.
The Third Aim i s to identify and characterize cytoplasmic molecules that interact with the P- selectin sorting signals. Cytoplasmic sorting molecules will be identified by: (a) co-immunoprecipitation of trans-Golgi network associated proteins with antibodies to P-selectin; (b) cross-linked or cytoplasmic domain peptides to trans-Golgi network membranes or cytosol; (c) preparation of anti-idiotype antibodies; (d) homology polymerase chain-reaction amplification from known adaptin sequences; and (e) expression cloning using the cytoplasmic domain of P-selectin as probe.
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