JBC Focus on PI3-Kinase with Echelon

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Stephens, D. J.
Right arrow Articles by Banting, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Stephens, D. J.
Right arrow Articles by Banting, G.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 274, Issue 42, 30080-30086, October 15, 1999


Direct Interaction of the trans-Golgi Network Membrane Protein, TGN38, with the F-actin Binding Protein, Neurabin*

David J. Stephens and George BantingDagger

From the Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TGN38 is a type I integral membrane protein that constitutively cycles between the trans-Golgi network (TGN) and plasma membrane. The cytosolic domain of TGN38 interacts with AP2 clathrin adaptor complexes via the tyrosine-containing motif (-SDYQRL-) to direct internalization from the plasma membrane. This motif has previously been shown to direct both internalization and subsequent TGN targeting of TGN38. We have used the cytosolic domain of TGN38 in a two-hybrid screen, and we have identified the brain-specific F-actin binding protein neurabin-I as a TGN38-binding protein. We demonstrate a direct 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 interaction is dependent on the serine (but not tyrosine) residue of the known TGN38 trafficking motif. We show that TGN38 interacts with the coiled coil region of neurabin in vitro and binds preferentially with the dimeric form of neurabin. TGN38 and neurabin also interact in vivo as demonstrated by coimmunoprecipitation from stably transfected PC12 cells. These data suggest that neurabin provides a direct physical link between TGN38-containing membranes and the actin cytoskeleton.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Many membrane proteins undergo complex trafficking itineraries within eukaryotic cells, cycling between intracellular compartments and the plasma membrane during such processes as nutrient uptake, biosynthetic protein sorting, intracellular signaling, and cell movement. In recent years, considerable efforts have been made to identify the individual components of these processes. Despite rapid progress in identifying the molecular components of the endocytic machinery (1), components of endoplasmic reticulum-to-Golgi transport pathways (2), and components of the exocytic machinery (3), little is known about the molecular machinery involved in post-TGN1 protein trafficking.

The type I membrane protein, TGN38, cycles between the TGN and cell surface via endosomal intermediates (4-7). Internalization from the plasma membrane and subsequent traffic back to the TGN is directed by the motif -SDYQRL- within the cytosolic domain of the protein (6, 8-10). We and others (11, 12) have shown that this protein is internalized from the plasma membrane in a clathrin-dependent manner through direct interaction with the µ2 subunit of the clathrin adaptor complex, AP2. Biochemical data have been obtained showing that formation of secretory vesicles at the TGN requires a protein complex that coimmunoprecipitates with TGN38 (13). This complex consists of a 62-kDa subunit with homology to phosphatidylinositol 3-kinase regulatory subunits and a 25-kDa GTPase in addition to TGN38 (13, 14). The small GTPase has been suggested to be Rab6 (13), but cDNA clones encoding p62 and this small GTPase have yet to be isolated.

Other protein complexes that function at a post-TGN level have also been identified. A considerable amount of data has been gathered regarding the role of a number of vacuolar protein sorting mutants in yeast. This work has led to the identification of a complex of proteins, conserved in higher eukaryotes, that is involved in endosome to Golgi transport (15, 16). Many individual components of trafficking pathways have also been identified from yeast two-hybrid screens using integral membrane protein cytosolic domains as bait. Examples include the identification of TIP47, which binds a specific motif within the cytosolic domain of the cation-independent mannose 6-phosphate receptor directing traffic from endosomes to the TGN (17), and P-CIP-1 (18), which mediates the endosomal trafficking of peptidylglycine alpha -amidating monooxygenase. The endoprotease furin has also been successfully used to identify novel components of the post-TGN sorting machinery. Two-hybrid screens of cDNA libraries using the cytosolic domain of furin as bait led to the identification of both PACS-1 (19) and the actin-binding protein ABP-280 (20) as molecules involved in trafficking of furin.

We chose to use the cytosolic domain of TGN38 to screen a two-hybrid library to identify interacting proteins, particularly those that may be involved in post-TGN membrane traffic. By using this approach we have identified a direct interaction between TGN38 and the brain-specific F-actin binding protein neurabin-I. We show that TGN38 also interacts directly with the ubiquitously expressed isoform of neurabin-I, neurabin-II (also known as spinophilin). These interactions are highly specific to TGN38 and can be abolished by a point mutation of a serine residue within the cytosolic domain of TGN38. TGN38 preferentially binds to the dimeric form of neurabin-I in vitro. The two proteins can also be coimmunoprecipitated from PC12 cells stably transfected with expression constructs encoding GFP-tagged neurabin-I or -II. These data suggest that neurabin provides a direct link between TGN38-containing membranes and the actin cytoskeleton.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

All reagents were purchased from Sigma unless otherwise stated. DNA restriction and modifying enzymes were from Roche Molecular Biochemicals.

Yeast Two-hybrid Screen-- The entire 33-amino acid cytosolic domain of TGN38 was cloned into the two-hybrid bait vector pBTM116 (21) to generate pBTM-TGN38 (12). This plasmid was then transformed into yeast strain L40 (21) as described previously (12). A 5-ml overnight culture of yeast containing the pBTM-TGN38 plasmid was grown in synthetic medium, Yc (1.2 g·liter-1 yeast nitrogen base without (NH4)2SO4, 5 g·liter-1 (NH4)2SO4, 10 g·liter-1 succinic acid, 6 g·liter-1 NaOH, 2% glucose) supplemented with 0.1 g·liter-1 adenine, arginine, cysteine, threonine, and leucine and 0.05 g·liter-1 aspartate, isoleucine, methionine, phenylalanine, proline, serine, tyrosine, and valine (i.e. lacking tryptophan, uracil, histidine, and lysine). This was then used to inoculate a 100-ml culture of the same medium that was then grown overnight and used to inoculate a 1000-ml culture of YPAD (YEPD (20 g·liter-1 peptone, 10 g·liter-1 yeast extract, pH 5.8, 2% glucose) with 40 µg·ml-1 adenine) that had been prewarmed to 30 °C. Cells were grown for 3 h, pelleted, washed in 500 ml of 1× TE (10 mM Tris·HCl, pH 7.5, 1 mM EDTA), and finally resuspended in 20 ml of 0.5× TE (5 mM Tris·HCl, pH 7.5, 0.5 mM EDTA) containing 100 mM lithium acetate. To this, 1.0 ml of 10 mg·ml-1 denatured and sheared salmon sperm DNA was added with 0.5 mg of library plasmid DNA (a rat brain cDNA library (kindly provided by Dr. Jeremy Henley, University of Bristol) consisting of both random-primed and oligo(dT)-primed cDNA from RNA of 100 pooled rat brains from 10- to 12-week-old Harlan Sprague-Dawley males, representing approximately 2,000,000 independent clones (CLONTECH, Basingstoke, UK)). After mixing, 140 ml of 1× TE containing 100 mM lithium acetate and 40% polyethylene glycol 3350 were added. This was thoroughly mixed and placed at 30 °C for 30 min, transferred to a sterile 2-liter beaker, and 17.6 ml of dimethyl sulfoxide added. This was then heat-shocked for 6 min at 42 °C with occasional mixing. Cells were then washed in a further 500 ml of 1× TE and resuspended in 1000 ml of YEPD. After a 1-h incubation at 30 °C with gentle mixing, the cells were washed in synthetic medium (Yc), lacking leucine, tryptophan, lysine, and uracil, before incubating overnight in 100 ml of the same. Cells were finally washed twice in 1× TE and resuspended in 10 ml of 1× TE before plating on selection medium (Yc containing 20 g·liter-1 agar, lacking tryptophan, leucine, histidine, lysine, and uracil). The entire transformation was split evenly between eight 22 × 22-cm square Petri dishes. Aliquots were also plated to determine transformation efficiency onto plates lacking leucine, lysine, tryptophan, and uracil. After 8 days growth at 30 °C, colonies were picked to duplicate plates with or without histidine and assayed as described in the Yeast Protocols Handbook (CLONTECH, Basingstoke, UK). Colonies that were scored double-positive for both histidine selection and beta -galactosidase were amplified in liquid culture, plasmid DNA-extracted, and library plasmids isolated by transformation into HB101 Escherichia coli. Plasmids were isolated by miniprep purification (Qiagen, Crawley, UK) and isolated library plasmids retransformed to L40 with the negative control plasmid, pLexA-Lamin. Those which were negative for interaction with lamin were analyzed as follows. Plasmids were sequenced using vector-specific primers (insert-screening amplimers, CLONTECH) using the DNA sequencing service within the Department of Biochemistry, University of Bristol. DNA sequences were used to search the non-redundant GenBankTM data base using the BLAST search algorithm available over the Internet from the European Bioinformatics Institute.

Plasmid Construction and Recombinant Protein Production-- Plasmids containing full-length cDNAs of neurabin-I and neurabin-II were obtained from Professor Yoshimi Takai (ERATO Biotimer Project, Kobe, Japan) and Professor Paul Greengard (The Rockefeller University, New York). The PstI fragment of neurabin-I containing the majority of the coiled coil domain (amino acids 719-1023) was subcloned to pRSETc (Invitrogen, Groningen, Netherlands) and expressed as a hexahistidine-tagged fusion protein in E. coli strain BLRDE3 (Novagen, Cambridge, UK) according to recommended protocols (Invitrogen). A fragment of neurabin-II (BamHI-EcoRI fragment containing amino acids 543-814) containing the majority of neurabin-II coiled coil regions was similarly expressed. A neurabin-II construct equivalent to the neurabin-I clone identified in the library screen (amino acids 483-817) was generated by PCR and cloned into pGAD10 (CLONTECH). Recombinant TGN38 cytosolic domain fusion proteins were generated as described previously (12). Other TGN38 constructs used in this study have also been previously described (12, 22). Other two-hybrid constructs were generated by PCR according to standard protocols.

The full coding sequence of neurabin-I was amplified by PCR and cloned into pEGFP-C1 (CLONTECH) according to standard protocols. The BglII restriction fragment of neurabin-II (amino acids 1-801, lacking the carboxyl-terminal 16 amino acids of neurabin-II) was subcloned to pEGFP-N3 (CLONTECH) generating an in-frame fusion with enhanced GFP (it was not found possible to amplify the full coding region of neurabin-II by PCR, presumably due to the high GC content of the 5' end of the cDNA).

Surface Plasmon Resonance Measurements-- Histidine-tagged and GST-tagged recombinant fusion proteins were expressed in BLRDE3 E. coli (Novagen) and purified according to standard protocols using Talon resin (CLONTECH) or glutathione-Sepharose (Sigma), respectively. Surface plasmon resonance analyses were performed on a BIAcore 1000 biosensor using phosphate-buffered saline as running buffer. Manufacturer's recommended immobilization protocols for histidine-tagged and GST-tagged fusion proteins were followed. Data were aligned using BIAcore evaluation software and prepared for publication using Microsoft Excel (Microsoft, Reading, UK).

Far Western Blotting-- Purified recombinant hexahistidine-tagged neurabin-I (coiled coil region) was separated by SDS-PAGE on an 8% gel, transferred to nitrocellulose (Schleicher & Schuell), and blocked overnight in TBS containing 0.05% Tween 20 with 10% dried skimmed milk (blocking buffer). The blot was then incubated with 50 µg·ml-1 Trx-TGN38 (a thioredoxin fusion of the entire cytosolic domain of TGN38, Ref. 12), washed (3 × 10 min) in TBS containing 0.05% Tween 20, and incubated with a 1:1000 dilution of an anti-thioredoxin polyclonal antibody (raised in rabbit using purified recombinant thioredoxin as immunogen, Ref. 23). The blot was washed again and incubated with a 1:10000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody (Sigma). Immunoreactive bands were visualized by chemiluminescence (Western blotting Kit, Roche Molecular Biochemicals).

Cell Culture, Transfection, and Cell Imaging-- PC12 cells (Ref. 24; a kind gift of Dr. Frank Gunn-Moore, University of Bristol) were cultured on plastic dishes in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 5% horse serum, penicillin, and streptomycin. Cells were transfected using Lipofectin (Life Technologies Inc.) according to standard protocols. Stable cell lines were selected using 400 µg·ml-1 G418 (Life Technologies Inc.) and maintained in 200 µg·ml-1 G418. For all cell imaging applications, cells were grown on collagen-coated glass coverslips. Immunofluorescence staining was performed according to established protocols (described in Ref. 25). The following antibodies were used: monoclonal anti-TGN38, clone 2F7.1 (26, Affinity Bioreagents, Golden, CO), anti- mannosidase-II (clone 53FC3, see Refs. 27 and 28). Primary antibodies were detected by incubation with 1:1000 dilutions of either Alexa-488- or Alexa-594-conjugated anti-mouse or anti-rabbit secondary antibodies (Molecular Probes, Cambridge, UK). Rhodamine-conjugated phalloidin was kindly provided by Professor Jeremy Tavaré (University of Bristol).

Coimmunoprecipitation-- Stably transfected PC12 cells expressing GFP-neurabin-II were grown to approximately 70% confluence in T75 culture flasks, lysed in RIPA-containing protease inhibitors as described previously (25) supplemented with 20 µg·ml-1 latrunculin B (Calbiochem). Cleared supernatants were incubated with 2 µl each of polyclonal anti-TGN38 antibodies, 1918 and G29 (29), or 4 µl of G29 preimmune serum, prebound to Gammabind (Amersham Pharmacia Biotech). After 2 h mixing at 4 °C, immunoprecipitates were washed six times (5 min each) in RIPA supplemented with protease inhibitors as for cell lysis. After boiling in SDS sample buffer, samples were separated on 6% polyacrylamide gels, transferred to nitrocellulose, and probed with 1:2000 anti-GFP polyclonal antibody (CLONTECH). Blots were developed as described above. Binding was competed by including 30 or 100 µM Trx-TGN38 (a fusion protein including the entire cytosolic domain of TGN38 (12)) as indicated.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Neurabin-I as a TGN38-binding Protein-- The entire cytosolic domain of TGN38 was used as bait in a screen of 6.5 × 106 clones of a rat brain cDNA library. This represented greater than 3-fold redundancy since the library contained 2 × 106 independent clones (see "Experimental Procedures"). 64 clones that showed positive interaction with TGN38 by both histidine autotrophy and expression of beta -galactosidase activity were isolated. Of these 64 clones, three represented a partial clone of the F-actin binding protein, neurabin-I (30). These clones (numbers 11, 17, and 118) were identical, showing 100% identity to amino acids 488-1095 of the published sequence of neurabin-I. This region of sequence includes the PSD-95/discs large/ZO-1 (PDZ) domain of neurabin-I as well as the entire carboxyl-terminal coiled coil region but is missing the amino-terminal actin binding domain. PDZ domains are known to interact with the extreme cytosolic domain of transmembrane receptors (31). However, a PDZ domain containing construct of neurabin-I (amino acids 488-718) showed no interaction with the cytosolic domain of TGN38 in our two-hybrid system, leading us to the conclusion that the PDZ domain of neurabin-I does not bind to TGN38.

We also tested the ability of the full coding region of neurabin-I to interact with TGN38. Upon prolonged incubation in either growth or beta -galactosidase assays, an interaction could be detected; the data showed that this interaction was significantly lower in affinity to that of the partial clone isolated from the library screen. We went on to characterize this interaction in more detail, screening library clone 118 against a number of TGN38 cytosolic domain constructs as well as a number of other receptor cytosolic domain constructs (all cloned as bait in pBTM116). Fig. 1 shows that as well as a strong positive interaction with the wild-type cytosolic domain of TGN38 (Fig. 1, B1), clone 118 showed a positive interaction with TGN38 in which the last four amino acids (NLKL) had been deleted (Fig. 1, E1). This further suggests that the interaction is not mediated through the PDZ domain of neurabin-I, since PDZ domains are believed to bind to the terminal residues of membrane protein cytosolic domains (31).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1.   Two-hybrid analysis of TGN38-neurabin interactions. Panels from left to right show transformants grown in the presence of histidine, transformants grown in the absence of histidine, and filter beta -galactosidase (beta-gal) assays, respectively. Colonies represent cotransformants with neurabin-I (rows 1 and 2) or neurabin-II (rows 3 and 4) and the following bait plasmids: A1 and A3, pLexA-Lamin (negative control); B1 and B3, pBTM-TGN38 (WT); C1 and C3, pBTM-TGN38 (Y333A); D1 and D3, pBTM-TGN38 (S331A); E1 and E3, pBTM-TGN38 (Delta NLKL); A2 and A4, pBTM-CD63; B2 and B4, pBTM-lgp120; C2 and C4, pBTM-trkB; D2 and D4, pBTM-pIgR; E2 and E4, pBTM-SIV Env. Positive signals are seen for each of neurabin-I and neurabin-II with pBTM-TGN38 (wild type, B1 and B3), pBTM-TGN38 (Y333A, C1 and C3), and pBTM-TGN38 (Delta NLKL, E1 and E3) only.

Neurabin-I was found to interact with a TGN38 construct in which the critical tyrosine residue of the internalization motif was mutated to alanine (Y333A). Mutation of serine 331 of TGN38 to alanine (S331A, Fig. 1, C1) completely abolished binding of neurabin-I (clone 118, Fig. 1, D1), an effect also seen for the interaction between TGN38 and the µ2 subunit of the AP2 clathrin adaptor (12). Together, these data suggest that neurabin and µ2 may have distinct but overlapping binding sites on the TGN38 cytosolic domain. The S331A mutant protein is functionally expressed in this system, since it interacts with another clone we have isolated in a different library screen.2 In addition we also see no interaction between TGN38 and neurabin-I in assays using TGN38 constructs in which serine 331 is mutated to aspartate (not shown). The specificity of interaction between TGN38 and neurabin-I is further demonstrated by the lack of detectable interaction in the two-hybrid system of clone 118 with the cytosolic domains of CD63, TrkB, Lgp120, Simian immunodeficiency virus envelope, or the polymeric immunoglobulin receptor (Fig. 1, row 2). A diagrammatic representation of the TGN38-neurabin interaction is shown in Fig. 2.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2.   Schematic representation of neurabin-I and neurabin-II and their interactions with TGN38, protein phosphatase I, and putative SH3 domain, and D2 dopamine receptor-binding sites. ABD, actin binding domain; black boxes represent coiled coil regions. Note that TGN38 preferentially binds to the dimeric form of neurabin-I in vitro (Fig. 4).

Analysis of TGN38-Neurabin-II Interactions-- Given the recent characterization of neurabin-II (spinophilin, Refs. 32 and 33), the ubiquitous homologue of neurabin-I, we decided to investigate the interaction of neurabin-II with TGN38. Owing to the weakly observed signal for interaction of TGN38 with the full coding sequence of neurabin-I in the two-hybrid system (which we also observed for neurabin-II (not shown)), we generated a neurabin-II clone that precisely corresponded to the neurabin-I clone isolated from the library screen (neurabin-II amino acids 483-817) and tested this truncated neurabin-II construct for interaction with the same panel of receptor cytosolic domain constructs described above. The results are shown in Fig. 1 (rows 3 and 4). Exactly the same pattern of interactions is observed for neurabin-II as was seen for neurabin-I. Whereas wild-type TGN38 interacts with neurabin-II (Fig. 1, B3), mutation of serine 331 to alanine abolishes this interaction (Fig. 1, D3). Mutation of tyrosine 333 to alanine or removal of the last four amino acids of TGN38 has no effect on this interaction (Fig. 1, C3 and E3). Similarly we detect no binding of neurabin-II to any of the other receptor cytosolic domains tested (Fig. 1, row 4). Since TGN38 is ubiquitously expressed (4), it is of great significance that an interaction with neurabin-II is detected as this is also expressed in all tissues examined (32, 33).

In Vitro Protein Interaction Assays-- To confirm that the interaction between TGN38 and neurabin is direct, we generated recombinant fusion proteins incorporating the cytosolic domain of TGN38 as a glutathione S-transferase (GST) fusion and the coiled coil domain of neurabins-I and -II as hexahistidine-tagged fusions. We reasoned from the two-hybrid data that the coiled coil domains of the neurabins were the most likely to interact with TGN38. Surface plasmon resonance experiments were performed to measure the interactions in real time. Fig. 3A shows the results of an experiment in which the neurabin-I fusion was immobilized onto a charged Ni2+-NTA biosensor chip (0-60 s) followed by direct injection of either purified GST (gray circles) or a GST-TGN38 fusion (black circles). The data show that a robust binding of the GST-TGN38 fusion is detected with no detectable binding of GST alone. These data confirm the interaction between TGN38 and neurabin-I identified from the two-hybrid screen. As with our two-hybrid data, we went on to measure binding of the coiled coil domain of neurabin-II to the cytosolic domain of TGN38. In this experiment, represented in Fig. 3B, we immobilized a GST-TGN38 fusion to the biosensor chip surface and flowed purified fusions of either neurabin-I (gray circles) or neurabin-II (black circles) coiled coil regions (amino acids 719-1023 and 543-814, respectively) across (note that this is the reciprocal of the previous experiment). As is evident from Fig. 3B, we detect almost identical binding of these fusions to TGN38, whereas we detect no binding to negative control surfaces (coated with GST alone, data not shown). As a further control experiment, we attempted to measure binding of tropomyosin to the GST-TGN38 fusion. Tropomyosin contains significant regions of predicted coiled coil structure (34) similar to those of neurabin-I and -II used in these experiments. The recent identification of tropomyosin isoforms on Golgi membranes (35) makes this a particularly suitable negative control for these TGN38 interaction assays. However, we detected no interaction of TGN38 with tropomyosin by surface plasmon resonance (not shown). We were also able to confirm the specificity of these interactions by generating fusion proteins in which the tyrosine 333 or serine 331 residues of TGN38 were mutated to alanine. As for our two-hybrid analysis, the mutation S331A abolished binding while mutation Y333A had no effect (not shown).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3.   Surface plasmon resonance analysis of TGN38 neurabin interactions. A, a sensor chip was loaded with (His)6-neurabin-I coiled coil fusion protein (0-120 s, marked with arrow 1 on panel A) after which either GST (gray circles) or GST-TGN38 (black circles) was flowed over the surface (300-600 s, arrow 2). Arrow 3 marks the start of the dissociation phase. B, recombinant fusions containing coiled coil domains of either neurabin-I (gray circles) or neurabin-II (black circles) were flowed over a sensor chip preloaded with GST-TGN38 bound to a covalently immobilized anti-GST antibody. Binding is monitored between 60 and 150 s (arrows 1 and 2). In both panels, binding is measured as an increase in resonance units on the y axis.

To analyze further the interaction between our recombinant fragments of neurabin and TGN38, we performed far Western blotting. Soluble neurabin-I coiled coil domain was separated by SDS-PAGE and blotted onto nitrocellulose. The soluble fraction obtained on expression of the coiled coil domain fragment of neurabin-I in E. coli is clearly resolved as monomeric, dimeric, and trimeric forms. This is consistent with the known capacity of coiled coil proteins to oligomerize. Following incubation with Trx-TGN38 cytosolic domain (12), the blot was developed using antibodies against the Trx fusion tag. The results of this analysis are shown in Fig. 4. Ponceau S staining of the membrane (Fig. 4, lane 2) shows that the predominant forms of the neurabin-I coiled coil domain expressed in this manner are dimeric and trimeric (monomeric neurabin-I is not visible by Ponceau S staining but migrates as shown by the arrow on the right of Fig. 4). Far Western blotting of these lanes shows that recombinant Trx-TGN38 cytosolic domain preferentially binds to the dimeric form of the neurabin-I coiled coil domain (Fig. 4, lane 4). It is of note that this is likely to be the naturally occurring form of the protein (30). No binding of Trx alone is seen (Fig. 4, lane 3).


View larger version (30K):
[in this window]
[in a new window]
  		Fig. 4.   Far Western blotting analysis of TGN38-neurabin interaction. Samples of recombinant neurabin-I coiled coil domains were separated by SDS-PAGE, stained with Ponceau S to visualize total protein (lanes 1 and 2), and subsequently probed with Trx alone (lane 3) or Trx-TGN38 fusion protein (lane 4). Binding was visualized using an anti-Trx polyclonal antibody and chemiluminescent detection. Positions of molecular mass standards (kDa) as visualized by Ponceau S staining are shown (lane 1).

Generation of Stable GFP-Neurabin-I and GFP-Neurabin-II PC12 Cell Lines-- In order to study the interaction of TGN38 with neurabin in vivo, we chose to use the rat pheochromocytoma cell line, PC12, which can be differentiated to a neuron-like morphology upon stimulation with nerve growth factor (24). Neurabin-I is exclusively expressed in brain (30), whereas neurabin-II is highly enriched in neuronal tissue (32, 33). Unfortunately, we were limited in this approach by a lack of specific antibodies to neurabin-I and -II, meaning we were unable to detect endogenous protein. Consequently, we stably transfected PC12 cells with GFP fusion constructs of neurabin-I or neurabin-II. To confirm that the GFP-tagged proteins were correctly localized, we performed confocal microscopy to examine the intracellular localization of these fusion proteins. These data (Fig. 5) show that both GFP-neurabin-I and GFP-neurabin-II localize to the actin cytoskeleton, partially but not completely overlapping with phalloidin staining (compare A with A' and D with D'). This is particularly apparent in areas of intense phalloidin staining at the periphery of the cell (such as neurite tips and other membrane projections). It is of note that the two patterns of fluorescence do not totally overlap and that actin labeling (as adjudged by phalloidin staining) is slightly more membrane proximal to neurabin localization. This is evident for both neurabin-I and neurabin-II. This may be a reflection of the currently undefined in vivo function of the neurabins.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 5.   Localization of GFP-neurabin-I and GFP-neurabin-II in stably transfected PC12 cells. Cells expressing GFP-neurabin-I (A, B, and C) or GFP-neurabin-II (D, E, and F) were fixed and stained with Rhodamine-phalloidin (A' and D'), anti-TGN38 (B' and E') or anti-mannosidase-II (C' and F'). Images shown are projections of multiple confocal sections throughout the entire depth of the cells. Bars, 5 µm.

We subsequently used confocal fluorescence microscopy to localize neurabin and TGN38 in the transfected PC12 cells. Neurabin-I and neurabin-II were localized by virtue of GFP fluorescence along with either TGN38 or mannosidase II (each visualized with specific antibodies). As is evident from Fig. 5 (panels B and B' and E and E'), there is no significant colocalization between the steady state distributions of TGN38 and either GFP-neurabin-I or GFP-neurabin-II. We could also not detect any gross changes in morphology or intracellular location of TGN38 immunoreactivity in the GFP-neurabin-transfected cells. In addition, upon investigation of the localization of mannosidase-II (a resident enzyme of the cis-, medial-Golgi cisternae (27, 28)), we observed no difference in mannosidase-II immunoreactivity compared with untransfected cells (Fig. 5, C and C' and F and F'). This further suggests that Golgi morphology is not compromised in these cells. It should be noted that those TGN38 molecules en route to or from the cell surface (see Ref. 36) are not detectable by immunofluorescence analysis using available antibodies (see "Discussion").

Coimmunoprecipitation Analyses-- Generation of these stable cell lines also enabled us to perform coimmunoprecipitation analysis using antibodies to TGN38 and GFP. Fig. 6 shows the results of such an experiment. Cells were lysed in the presence of latrunculin B (in order to depolymerize the actin cytoskeleton) and TGN38-immunoprecipitated. These immunoisolated samples were then separated by SDS-PAGE and blotted onto nitrocellulose membrane. Blots were developed with an anti-GFP polyclonal antibody to detect specifically immunoprecipitated neurabin fusion proteins. As is shown in Fig. 6, this protocol led to the detection of GFP-neurabin-I. Proteins of molecular masses between 120 and 160 kDa were visualized (Fig. 6, lane 2). Multiple bands were consistently visualized suggesting that GFP-neurabin-I is readily degraded in these cells. The anti-GFP polyclonal antibody failed to detect anything by immunoblot when irrelevant polyclonal antibodies (preimmune rabbit serum) were used in the immunoprecipitation step of this procedure (Fig. 6, lane 1) confirming the specificity of interaction of neurabin with TGN38. Furthermore, we were able to inhibit the TGN38-neurabin interaction in a concentration-dependent manner by including a TGN38 cytosolic domain fusion protein in the immunoprecipitation assays (Fig. 6, lanes 3 and 4 and data not shown). Identical results were obtained using lysates from cells expressing GFP-neurabin-II (not shown) and in experiments using monoclonal anti-GFP antibodies or polyclonal anti-neurabin-II antibody in the immunoblotting step (data not shown). Furthermore, using anti-GFP antibodies, we have been able to specifically immunoprecipitate TGN38 from GFP-neurabin-I and GFP-neurabin-II PC12 cell lysates.


View larger version (69K):
[in this window]
[in a new window]
  		Fig. 6.   Coimmunoprecipitation of GFP-neurabin-I with TGN38. PC12 cells stably expressing GFP-neurabin-I were grown to 70% confluence, lysed in the presence of latrunculin B, and immunoprecipitated with preimmune serum (lane 1) or anti-TGN38 antibodies (lanes 2, 3, and 4). Interaction was competed by addition of Trx-TGN38 fusion protein to the immunoprecipitation reactions at a final concentration of 30 (lane 3) or 100 µM (lane 4). After washing, immune complexes were separated on 6% polyacrylamide gels, blotted to nitrocellulose, and probed with a polyclonal anti-GFP antibody. Positions of molecular mass standards (kDa) are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The actin cytoskeleton is responsible for many central functions of eukaryotic cells from the maintenance and regulation of cell shape to cell motility and intracellular transport. It mediates several essential roles during mitosis (37); together with adhesion molecules, it regulates cell-cell and cell-substrate interactions (38, 39) and participates in transmembrane signaling (40), endocytosis (41, 42), and secretion (43). Actin filament integrity has been shown to be required for the normal positioning and morphology of the Golgi complex (44). Myosin family motor proteins regulate vesicle budding and traffic from the Golgi (45-48), and recent data also implicate the actin-binding proteins tropomyosin 5 and centractin in Golgi function (35).

We have identified the F-actin binding protein neurabin as a direct link between the integral membrane protein TGN38 and actin filaments. We have shown that these proteins interact in two-hybrid assays, in vitro and in vivo. In our two-hybrid screen we identified a partial clone of neurabin-I as a TGN38-interacting protein. This 1821-base pair clone lacks the amino-terminal actin binding domain of neurabin but includes the entire predicted PDZ domain as well as the carboxyl-terminal coiled coil regions (30). Interaction between the TGN38 cytosolic domain and full-length neurabin is barely detectable in these assays. This can be explained in a number of ways. First, the presence of the actin binding domain may result in sequestration of the two-hybrid fusion protein to actin filaments in the yeast cells used for these assays, thereby precluding transport to the nucleus. Alternatively, it is possible that incorporation of the actin binding domain generates a fusion protein in which, owing to the extended coiled coil structure of neurabin, the amino-terminal trans-activating domain is at such a distance from the TGN38 binding region as to preclude reconstitution of an active transcription factor. It should be noted that the length of neurabin-I clones isolated is consistent with the average insert size of the library (1.2 kilobase pairs). We further demonstrated in our two-hybrid system that TGN38 is capable of interacting with neurabin-II, the ubiquitously expressed isoform of neurabin. This is of particular relevance since the expression pattern of neurabin-II reflects that of TGN38.

We have been unable to demonstrate colocalization of TGN38 with neurabin-I or -II at the level of light microscopy, but it should be noted that we are only able to visualize a subset of TGN38 in these cells, namely that which is localized to the TGN. TGN38 is a highly dynamic protein within the cell, rapidly cycling between the TGN and plasma membrane via endosomal intermediates (reviewed in Ref. 36). It will be interesting to determine the way in which interaction with neurabin modulates the known intracellular trafficking itinerary of TGN38. There are a number of possibilities for the role of the interaction between TGN38 and neurabin given the evidence for roles of the actin cytoskeleton in budding from the TGN (53), endocytosis (41, 42), and recycling from endocytic compartments to the plasma membrane (41, 42). It is possible that neurabin acts in competition with the AP2 endocytic adaptor complex (with which TGN38 also interacts, Ref. 12) to provide an intricate regulation of intracellular versus plasma membrane-localized TGN38.

From our analysis of TGN38 mutant constructs, we have determined that interaction with neurabin is entirely dependent on the serine residue that lies within the known intracellular sorting motif of TGN38, SDYQRL. Mutation of this serine residue to alanine or aspartate (both of which abolish the interaction with neurabins-I and -II) significantly alters the intracellular trafficking of TGN38, leading to a decreased efficiency of sorting back to the TGN from endosomal compartments and concomitant increase in lysosomal degradation of the protein (25). We are currently attempting to address whether these observed trafficking defects of S331A TGN38 are due directly to a lack of interaction with neurabin.

We have so far not found any alterations in either steady state localization or endocytosis from the plasma membrane of TGN38 upon overexpression of neurabin-I or neurabin-II. It remains possible that neurabin directly facilitates trafficking of TGN38-containing membranes through further interactions with motor proteins, presumably belonging to the myosin superfamily. Indeed, we have data showing highly dynamic movement of GFP-neurabin-II in cultured PC12 cells supporting this possibility.3 Interestingly, the PDZ domain containing protein, TACIP18, has recently been shown, through a direct interaction, to mediate the targeting of pro-transforming growth factor-alpha to the cell surface (54).

Neurabin-I provides a direct link between the actin cytoskeleton and the 70-kDa isoform of S6 kinase (p70S6k, (55)) thereby modulating p70S6k activity. Immunoisolated p70S6k is not capable of phosphorylating the cytosolic domain of TGN38 in vitro.4 The consensus motif for protein phosphatase 1 binding in neurabin-II (32) is also present in neurabin-I. There are also two potential tyrosine phosphorylation sites in both neurabin-I and -II which lie within the coiled coil regions, whereas two further potential tyrosine phosphorylation sites lie within a region adjacent to the PDZ domain of the neurabins. It is tempting to speculate that phosphorylation events may regulate binding of proteins such as p70S6k (55) and TGN38 to the neurabins. The potential for tyrosine (56) and/or serine (57) phosphorylation of TGN38 itself also provides a further level of potential regulation of these interactions.

Both neurabin-I and neurabin-II (30, 32, 33), like ABP-280 (20), are capable of cross-linking actin filaments. Thus, these proteins may represent a general mechanism for directly linking membranes to the actin cytoskeleton through direct interaction of actin-binding proteins with intracellular membranes. It is possible that when bound to neurabin, TGN38 will be immobilized to the actin cytoskeleton as is thought to occur on interaction of furin with ABP-280 (20). Diverse membrane proteins such as the beta 2 integrin, CD18, the prohormone processing enzyme, furin, the high affinity immunoglobulin receptor, Fcgamma RI, and the von Willebrand factor receptor complex all bind directly to ABP-280 (58-60). Interestingly, the steady state localizations of ABP280 (58) and furin (61) do not significantly overlap despite convincing demonstration of a physiologically relevant interaction between these two proteins (20).

Recent data showing a direct interaction between the third cytosolic loop of the D2 dopamine receptor and neurabin-II (spinophilin, see Ref. 62) are also of particular relevance given our findings. This interaction was found to be specific to neurabin-II and was not seen with neurabin-I. This suggests that neurabin-I and -II may have distinct as well as overlapping roles within eukaryotic cells. The presence of a sterile alpha motif domain in neurabin-I but not neurabin-II further emphasizes this point. The site of interaction of the D2 dopamine receptor with neurabin-II was identified between the actin binding and PDZ domains of neurabin-II (62), distinct from the TGN38-binding site (which we have mapped to the coiled coil region of neurabin-I and -II). This is entirely consistent with a possible role of neurabin as a molecular scaffold, providing a direct link between membranes and the actin cytoskeleton through direct, and potentially simultaneous, interactions with a number of transmembrane receptors.

    ACKNOWLEDGEMENTS

We thank the Medical Research Council for providing Infrastructure Award G4500006 to establish the School of Medical Sciences Cell Imaging Facility. We thank Dr. Mark Jepson and Alan Leard for assistance with cell imaging applications. We are also extremely grateful to Professor Yoshimi Takai, Dr. Hiroyuki Nakanishi, and Prof. Paul Greengard for the kind gifts of neurabin-I and -II cDNAs; Prof. Jeremy Tavaré, Drs. Paul Luzio, Benjamin Aroeti, Mark Marsh, and Barbara Reaves for the kind gifts of cDNA constructs and reagents; Dr. Jeremy Henley for the rat brain two-hybrid library, and Dr. Frank Gunn-Moore for providing PC12 cells. We are also particularly grateful to Drs. Sharon Milgram and Donelson Smith (University of North Carolina, Chapel Hill, NC) for sharing reagents and communicating results prior to publication.

    FOOTNOTES

* This work was supported by Medical Research Council and Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Tel.: 44 117 928 8272; Fax: 44 117 928 8274; E-mail: g.banting@bristol.ac.uk.

2 C. M. Crump, D. J. Stephens, and G. Banting, unpublished observations.

3 D. J. Stephens and G. Banting, submitted for publication.

4 D. J. Stephens and G. Banting, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: TGN, trans-Golgi network; GFP, green fluorescent protein; GST, glutathione S-transferase; PDZ, PSD95/discs large/ZO-1; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Trx, thioredoxin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Schmid, S. L. (1997) Annu. Rev. Biochem. 66, 511-548[CrossRef][Medline] [Order article via Infotrieve]
2. Rothman, J. E., and Wieland, F. T. (1997) Science 272, 227-234[Abstract]
3. Hsu, S.-C., Hazuka, C. D., Foletti, D. L., and Scheller, R. H. (1999) Trends Cell Biol. 9, 150-153[CrossRef][Medline] [Order article via Infotrieve]
4. Luzio, J. P., Brake, B., Banting, G., Howell, K. E., Braghetta, P., and Stanley, K. K. (1990) Biochem. J. 270, 97-102[Medline] [Order article via Infotrieve]
5. Reaves, B., Wilde, A., and Banting, G. (1992) Biochem. J. 283, 313-316
6. Reaves, B., Horn, M., and Banting, G. (1993) Mol. Biol. Cell 4, 93-105[Abstract]
7. Ponnambalam, S., Rabouille, C., Luzio, J. P., Nilsson, T., and Warren, G. (1994) J. Cell Biol. 125, 253-268[Abstract/Free Full Text]
8. Bos, K., Wraight, C., and Stanley, K. K. (1993) EMBO J. 12, 2219-2228[Medline] [Order article via Infotrieve]
9. Wong, S. H., and Hong, W. (1993) J. Biol. Chem. 268, 22853-22862[Abstract/Free Full Text]
10. Humphrey, J. S., Peters, P. J., Yuan, L. C., and Bonifacino, J. S. (1993) J. Cell Biol. 120, 1123-1135[Abstract/Free Full Text]
11. Ohno, H., Stewart, J., Fournier, M.-C., Bosshart, H., Rhee, I., Miyatake, S., Saito, T., Gallusser, A., Kirchhausen, T., and Bonifacino, J. S. (1995) Science 269, 1872-1875[Abstract/Free Full Text]
12. Stephens, D. J., Crump, C. M., Clarke, A. R., and Banting, G. (1997) J. Biol. Chem. 272, 14104-14109[Abstract/Free Full Text]
13. Jones, S. M., Crosby, J. R., Salamero, J., and Howell, K. E. (1993) J. Cell Biol. 122, 775-788[Abstract/Free Full Text]
14. Jones, S. M., and Howell, K. E. (1997) J. Cell Biol. 139, 339-349[Abstract/Free Full Text]
15. Seaman, M. N., Marcusson, E. G., Cereghino, J. L., and Emr, S. D. (1997) J. Cell Biol. 137, 79-92[Abstract/Free Full Text]
16. Seaman, M. N., McCaffery, J. M., and Emr, S. D. (1998) J. Cell Biol. 142, 665-681[Abstract/Free Full Text]
17. Diaz, E., and Pfeffer, S. R. (1998) Cell 93, 433-443[CrossRef][Medline] [Order article via Infotrieve]
18. Chen, L., Johnson, R. C., and Milgram, S. L. (1998) J. Biol. Chem. 273, 33524-33532[Abstract/Free Full Text]
19. Wan, L., Molloy, S. S., Thomas, L., Liu, G., Xiang, Y., Rybak, S. L., and Thomas, G. (1998) Cell 94, 205-216[CrossRef][Medline] [Order article via Infotrieve]
20. Liu, G., Thomas, L., Warren, R. A., Enns, C. A., Cunningham, C., Hartwig, J. H., and Thomas, G. (1997) J. Cell Biol. 139, 1719-1733[Abstract/Free Full Text]
21. Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A. (1993) Cell 74, 205-214[CrossRef][Medline] [Order article via Infotrieve]
22. Stephens, D. J., and Banting, G. (1998) Biochem. J. 335, 567-572
23. Crump, C. M., Williams, J. L., Stephens, D. J., and Banting, G. (1998) J. Biol. Chem. 273, 28073-28077[Abstract/Free Full Text]
24. Greene, L. A., and Tischler, A. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2424-2428[Abstract/Free Full Text]
25. Roquemore, E. P., and Banting, G. (1998) Mol. Biol. Cell 9, 2125-2144[Abstract/Free Full Text]
26. Horn, M., and Banting, G. (1994) Biochem. J. 301, 69-73
27. Baron, M. D., and Garoff, H. (1990) J. Biol. Chem. 265, 19928-19931[Abstract/Free Full Text]
28. Burke, B., Griffiths, G., Reggio, H., Louvard, D., and Warren, G. (1982) EMBO J. 1, 1621-1628[Medline] [Order article via Infotrieve]
29. Wilde, A., Reaves, B., and Banting, G. (1992) FEBS Lett. 313, 235-238[CrossRef][Medline] [Order article via Infotrieve]
30. Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A., and Takai, Y. (1997) J. Cell Biol. 139, 951-961[Abstract/Free Full Text]
31. Saras, J., and Heldin, C. H. (1996) Trends Biochem. Sci. 21, 455-458[CrossRef][Medline] [Order article via Infotrieve]
32. Allen, P. B., Ouimet, C. C., and Greengard, P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9956-9961[Abstract/Free Full Text]
33. Satoh, A., Nakanishi, H., Obaishi, H., Wada, M., Takahashi, K., Satoh, K., Hirao, K., Nishioka, H., Hata, Y., Mizoguchi, A., and Takai, Y. (1998) J. Biol. Chem. 273, 3470-3475[Abstract/Free Full Text]
34. Whitby, F. G., Kent, H., Stewart, F., Stewart, M., Xie, X., Hatch, V., Cohen, C., and Phillips, G. N., Jr. (1992) J. Mol. Biol. 227, 441-448[CrossRef][Medline] [Order article via Infotrieve]
35. Heimann, K., Percival, J. M., Weinberger, R., Gunning, P., and Stow, J. L. (1999) J. Biol. Chem. 274, 10743-10750[Abstract/Free Full Text]
36. Banting, G., and Ponnambalam, S. (1997) Biochim. Biophys. Acta 1355, 209-217[Medline] [Order article via Infotrieve]
37. Salmon, E. D. (1989) Curr. Opin. Cell Biol. 1, 89-93
38. Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62[CrossRef][Medline] [Order article via Infotrieve]
39. Luna, E. J., and Hitt, A. L. (1992) Science 258, 955-964[Abstract/Free Full Text]
40. Juliano, R. L., and Haskill, S. (1993) J. Cell Biol. 120, 577-585[Free Full Text]
41. Lamaze, C., Fujimoto, L. M., Yin, H. L., and Schmid, S. L. (1997) J. Biol. Chem. 272, 20332-20335[Abstract/Free Full Text]
42. Durrbach, A., Louvard, D., and Coudrier, E. (1996) J. Cell Sci. 109, 457-465[Abstract]
43. Kuznetsov, S. A., Langford, G. M., and Weiss, D. G. (1992) Nature 356, 722-725[CrossRef][Medline] [Order article via Infotrieve]
44. Valderrama, F., Babiá, T., Ayala, I., Kok, J. W., Renau-Piqueras, J., and Egea, G. (1998) Eur. J. Cell Biol. 76, 9-17[Medline] [Order article via Infotrieve]
45. Fath, K. R., Trimbur, G. M., and Burgess, D. R. (1994) J. Cell Biol. 126, 661-675[Abstract/Free Full Text]
46. Fath, K. R., and Burgess, D. R. (1995) J. Cell Biol. 120, 117-127[Abstract/Free Full Text]
47. Stow, J. L., Fath, K. R., and Burgess, D. R. (1998) Trends Cell Biol. 8, 138-141[CrossRef][Medline] [Order article via Infotrieve]
48. Stow, J. L., and Heimann, K. (1998) Biochim. Biophys. Acta 1404, 161-171[Medline] [Order article via Infotrieve]
49. Deleted in proof
50. Deleted in proof
51. Deleted in proof
52. Deleted in proof
53. Müsch, A., Cohen, D., and Rodriguez-Boulan, E. (1997) J. Cell Biol. 138, 291-306[Abstract/Free Full Text]
54. Fernandez-Larrea, J., Merlos-Suárez, A., Ureña, J. M., Baselga, J., and Arribas, J. (1999) Mol. Cell 3, 423-433[CrossRef][Medline] [Order article via Infotrieve]
55. Burnett, P. E., Blackshaw, S., Lai, M. M., Qureshi, I. A., Burnett, A. F., Sabatini, D. M., and Snyder, S. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8351-8356[Abstract/Free Full Text]
56. Stephens, D. J., and Banting, D. J. (1997) FEBS Lett. 416, 27-29[CrossRef][Medline] [Order article via Infotrieve]
57. Zehavi-Feferman, R., Burgess, J. W., and Stanley, K. K. (1995) FEBS Lett. 368, 122-124[CrossRef][Medline] [Order article via Infotrieve]
58. Sharma, C. P., Ezzell, R. M., and Arnaout, M. A. (1995) J. Immunol. 154, 3461-3470[Abstract]
59. Ohta, Y., Stossel, T. P., and Hartwig, J. H. (1991) Cell 67, 275-282[CrossRef][Medline] [Order article via Infotrieve]
60. Meyer, S. C., Zuerbig, S., Cunningham, C. C., Hartwig, J. H., Bissel, T., Gardner, K., and Fox, J. E. B. (1997) J. Biol. Chem. 272, 2914-2919[Abstract/Free Full Text]
61. Molloy, S. S., Thomas, L., Van Slyke, J. K., Stenberg, P. E., and Thomas, G. (1994) EMBO J. 13, 18-33[Medline] [Order article via Infotrieve]
62. Smith, F. D., Oxford, G. S., and Milgram, S. L. (1999) J. Biol. Chem. 274, 19894-19900[Abstract/Free Full Text]

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Biol. CellHome page
T. Kimura, P. B. Allen, A. C. Nairn, and M. J. Caplan
Arrestins and Spinophilin Competitively Regulate Na+,K+-ATPase Trafficking through Association with a Large Cytoplasmic Loop of the Na+,K+-ATPase
Mol. Biol. Cell, November 1, 2007; 18(11): 4508 - 4518.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
D. Orioli, I. N. Colaluca, M. Stefanini, S. Riva, C. G. Dotti, and F. A. Peverali
Rac3-induced Neuritogenesis Requires Binding to Neurabin I
Mol. Biol. Cell, May 1, 2006; 17(5): 2391 - 2400.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
K. Venkateswarlu, T. Hanada, and A. H. Chishti
Centaurin-{alpha}1 interacts directly with kinesin motor protein KIF13B
J. Cell Sci., June 1, 2005; 118(11): 2471 - 2484.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
R. T. Terry-Lorenzo, D. W. Roadcap, T. Otsuka, T. A. Blanpied, P. L. Zamorano, C. C. Garner, S. Shenolikar, and M. D. Ehlers
Neurabin/Protein Phosphatase-1 Complex Regulates Dendritic Spine Morphogenesis and Maturation
Mol. Biol. Cell, May 1, 2005; 16(5): 2349 - 2362.
[Abstract] [Full Text] [PDF]