GAIP, a Galpha i-3-binding protein, is associated with Golgi-derived vesicles and protein trafficking

Proteins of theregulators of G protein signaling (RGS) family bind to G subunits todownregulate their signaling in a variety of systems. G-interactingprotein (GAIP) is a mammalian RGS protein that shows high affinity forthe activated state of G_i-3, aprotein known to regulate post-Golgi trafficking of secreted proteins in kidney epithelial cells. This study aimed to localize GAIP inepithelial cells and to investigate its potential role in the regulation of membrane trafficking.LLC-PK₁ cells were stably transfected with a c-myc-tagged GAIPcDNA. In the transfected and untransfected cells, GAIP was found in thecytosol and on cell membranes. Immunogold labeling showed thatmembrane-bound GAIP was localized on budding vesicles around Golgistacks. When an in vitro assay was used to generate vesicles fromisolated rat liver and Madin-Darby canine kidney cell Golgi membranes, GAIP was found to be concentrated in fractions of newly budded Golgivesicles. Finally, the constitutive trafficking and secretion ofsulfated proteoglycans was measured in cell lines overexpressing GAIP.We show evidence for GAIP regulation of secretory trafficking beforethe level of the trans-Golgi networkbut not in post-Golgi secretion. The location and functional effects ofGAIP overlap only partially with those ofG_i-3 and suggest multiple roles for GAIP in epithelial cells.