The transmembrane domain enhances granular targeting of P-selectin.
P-selectin is an integral membrane glycoprotein that is stored in granules of endothelial cells and platelets. The cytoplasmic domain of P-selectin is known to contain at least part of the signal that directs the protein to storage granules. In order to more fully understand how P-selectin is targeted to the regulated secretory pathway, we have expressed chimeric constructs between P- and E-selectin, a protein which is expressed on the cell surface, in a rat insulinoma cell line. Immunofluorescence studies indicated that replacing the cytoplasmic domain of E-selectin with that of P-selectin resulted in low-level granular expression. In contrast, when both the transmembrane and cytoplasmic domains of E-selectin were replaced with the analogous domains of P-selectin, the granular localization appeared greatly increased. This was confirmed by immunoelectron microscopy which demonstrated a three- to fourfold improvement in granular targeting, i.e. similar to wild-type P-selectin. The transmembrane domain had to be in the context of the P-selectin cytoplasmic domain as this membrane-spanning region could not induce granular targeting on its own. These results describe a novel function for the transmembrane domain of P-selectin in enhancing the efficiency of granular targeting and further implicate protein transmembrane domains in intracellular trafficking.
Fleming
J C
JC
Program in Cell, Molecular and Developmental Biology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA, USA.
Berger
G
G
Guichard
J
J
Cramer
E M
EM
Wagner
D D
DD
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D019040:E-Selectin / Q000096:biosynthesis / Q000235:genetics / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D007424:Intracellular Fluid / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D019007:P-Selectin / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000648:ultrastructure; D014162:Transfection; D014407:Tumor Cells, Cultured
D019040:E-Selectin; D019007:P-Selectin; D011993:Recombinant Fusion Proteins