Direct interaction of the trans-Golgi network membrane protein, TGN38, with the F-actin binding protein, neurabin

TGN38 is a type I integral membrane protein that constitutively cycles betw een the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-co ntaining motif (-SDYQRL-) to direct internalization from the plasma membran e, This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN 38 in a two-hybrid screen, and we have identified the brain-specific F-acti n binding protein neurabin-I as a TGN38-binding protein. We demonstrate a d irect interaction between TGN38 and the ubiquitous homologue of neurabin-I, neurabin-II (also called spinophilin), We have used a combination of yeast two-hybrid and in vitro protein interaction assays to show that this inter action is dependent on the serine (but not tyrosine) residue of the known T GN38 trafficking motif, We shaw that TGN38 interacts with the coiled coil r egion of neurabin in vitro and binds preferentially with the dimeric form o f neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coi mmunoprecipitation from stably transfected PC12 cells, These data suggest t hat neurabin provides a direct physical link between TGN38-containing membr anes and the actin cytoskeleton.