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First published online 18 October 2005
doi: 10.1242/jcs.02620

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NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS

Ann Icking^*, Simone Matt^*, Nils Opitz, Anja Wiesenthal, Werner Müller-Esterl and Kirstin Schilling

Institute for Biochemistry II, University of Frankfurt Medical School, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany

View larger version (16K): [in a new window]   Fig. 1. NOSTRIN belongs to the PCH family of proteins. (A) Domain structure of selected members of the human PCH protein family. The proteins share a characteristic composition comprising an N-terminal FCH domain, a C-terminal SH3 domain and a coiled-coil domain located C-terminally of the FCH domain. Some members including NOSTRIN, FBP17, CIP4 and Toca-1 also contain an HR1 motif, which harbors a coiled-coil domain and has been described as a Rho GTPase binding unit. (B) Phylogenetic tree of PCH family proteins. The phylogenetic tree was constructed by alignment of sequence data of denoted proteins (ClustalW), applying the Phylodraw0.8 software. According to this, pacsins represent the closest relatives of NOSTRIN.

View larger version (31K): [in a new window]   Fig. 2. NOSTRIN is able to self-associate. (A) Immobilized (His)6-tagged NOSTRIN was incubated with purified GST-NOSTRIN, or GST as control. Matrix-bound proteins were eluted and analyzed by immunoblotting using anti-GST and anti-(His)6. (B) NOSTRIN constructs used in the yeast two-hybrid assay. (C) A yeast mating assay was performed using the Matchmaker system (Clontech). Interaction of proteins was judged by growth and blue staining on 4x deficient plates (-His, -Ura, -Trp, -Leu) containing X-Gal.

View larger version (34K): [in a new window]   Fig. 3. NOSTRIN trimerizes in vitro and in vivo. (A) Gel filtration of purified recombinant GST-NOSTRIN242-506. Fractions were collected and analyzed by SDS-PAGE and western blotting using anti-GST. (B,C) Lysates of COS cells transiently transfected with Myc/(His)6-tagged full-size NOSTRIN or VSV-tagged NOSTRIN250-434 and NOSTRIN433-506, respectively, were treated with 0.3 mM DSS or mock-treated for control. Samples were analyzed by western blotting using anti-Myc or anti-VSV, respectively. The asterisks mark non-identified complexes of NOSTRIN.

View larger version (29K): [in a new window]   Fig. 4. NOSTRIN, by means of its SH3 domain, interacts with multiple proteins. (A) GST-NOSTRIN was used in a pull-down assay using lysates of CHO-eNOS cells. GST alone served as control. (B) Co-immunoprecipitation from CHO-eNOS cells infected with SFV-NOSTRIN-GFP or SFV-GFP (control), using anti-GFP. (C) Lysates of COS cells transfected with dynamin cDNA for 24 hours (upper panel) or untransfected (lower) were subjected to precipitation with GST-NOSTRIN, GST-NOSTRIN-SH3, GST-NOSTRIN-SH3 or GST alone. (D) Immobilized GST-NOSTRIN and GST were incubated with lysates of COS cells expressing wild-type dynamin (wt) or the dominant-negative mutant dynamin-K44A (K44A). Empty vector (-) served as control.

View larger version (120K): [in a new window]   Fig. 5. Dynamin and eNOS are assembled into common structures by NOSTRIN in a process requiring its SH3 domain. CHO-eNOS cells stably expressing eNOS were transiently transfected with cDNA encoding NOSTRIN (A-D), dynamin-2-GFP (E-H), NOSTRIN and dynamin-2-GFP (I-L), or NOSTRIN-SH3 and dynamin-2-GFP (M-P). Following fixation, cells were stained with anti-NOSTRIN (B,J,N) and anti-eNOS (C,G,K,O). (E,I,M) Fluorescence of GFP-fused dynamin-2. (D,H,L,P) Merged images of each row. (A-D) NOSTRIN and eNOS co-localize at vesicular and elongated structures throughout the cytoplasm. (E-H) In the absence of NOSTRIN, eNOS and dynamin-2-GFP hardly overlap. (I-L) Upon co-expression of dynamin-2-GFP and NOSTRIN, dynamin and eNOS are brought together at common structures. (M-P) NOSTRIN-SH3 does not affect localization of either eNOS or dynamin-2-GFP. Bars, 10 µm.

View larger version (104K): [in a new window]   Fig. 6. NOSTRIN, eNOS and N-WASP co-localize at actin filaments. CHO-eNOS cells were transiently transfected with cDNA encoding NOSTRIN (A-D), N-WASP-GFP (E-H), or NOSTRIN and N-WASP-GFP (I-P). Following fixation, cells were stained with anti-NOSTRIN (B,J,N), anti-eNOS (C,G,K) and phalloidin (O). (E,I,M) Fluorescence of GFP-fused N-WASP. (D,H,L,P) Merged images of each row. (A-D) Upon moderate expression, NOSTRIN localizes to vesicular and filamentous structures, where it co-localizes with eNOS. (E-H) When N-WASP-GFP is expressed alone, it is mainly diffusely dispersed in the cytoplasm and does not co-localize with eNOS. (I-L) Co-expression with NOSTRIN brings N-WASP-GFP to eNOS-positive, mostly filamentous structures. (M-P) NOSTRIN and N-WASP-GFP co-localize at structures running along phalloidin-labeled actin filaments. Bars, 10 µm.

View larger version (86K): [in a new window]   Fig. 7. NOSTRIN-dependent translocation of eNOS requires an intact actin cytoskeleton. CHO-eNOS cells infected with SFV encoding NOSTRIN for 8 hours were treated with cytochalasin D within the last 2 hours of infection (D-F) or mock-treated (A-C) as control. After fixation, cells were stained with anti-NOSTRIN (B,E) and anti-eNOS (A,D). NOSTRIN and eNOS co-localize in intracellular vesicle-like structures of untreated cells (A-C), whereas they accumulate in large patches in the periphery of cytochalasin-treated cells (arrows) (D-F). Bars, 10 µm.

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