FEBS Letters 583 (2009) 49–54

journal homepage: www.FEBSLetters .org

Interaction of the human somatostatin receptor 3 with the multiple PDZdomain protein MUPP1 enables somatostatin to control permeabilityof epithelial tight junctions

Chong Wee Liew a,1,4, Matthias Vockel b,d,4, Günter Glassmeier c, Johanna M. Brandner d,Gregorio J. Fernandez-Ballester e,2, Jürgen R. Schwarz c, Stefan Schulz f, Friedrich Buck a, Luis Serrano e,3,Dietmar Richter a, Hans-Jürgen Kreienkamp a,b,*

a Institut für Zellbiochemie und klinische Neurobiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germanyb Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germanyc Institut für Angewandte Physiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germanyd Klinik und Poliklinik für Dermatologie und Venerologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germanye Computational Biology Programme, EMBL, 69117 Heidelberg, Germanyf Institut für Pharmakologie und Toxikologie, Friedrich-Schiller-Universität Jena, 07743 Jena, Germany

a r t i c l e i n f o a b s t r a c t

Article history:Received 26 September 2008Revised 12 November 2008Accepted 18 November 2008Available online 9 December 2008

Edited by Beat Imhof

Keywords:Heterotrimeric G-proteinPDZ domainTight junction

0014-5793/$34.00 � 2008 Federation of European Biodoi:10.1016/j.febslet.2008.11.048

Abbreviations: GIRK, G-protein gated inwardly reGPCR, G-protein coupled receptor; MDCK, Madin-Dmultiple PDZ domain protein 1; PDZ, PSD-95/discs lartic density protein of 95 kDa; TER, transepithelial reSST, somatostatin; SSTR, somatostatin receptor; ZO-1

* Corresponding author. Fax: + 49 40 42803 5138.E-mail address: Kreienkamp@uke.uni-hamburg.de

1 Present address: Joslin Diabetes Center, Harvard MeBoston, MA 02215, USA.

2 Present address: IBMC, Universidad Miguel Hernan3 Present address: Systems Biology, Center for Genom4 These authors contributed equally to this manuscr

The presence of heterotrimeric G-proteins at epithelial tight junctions suggests that these cellularjunctions are regulated by so far unknown G-protein coupled receptors. We identify here an inter-action between the human somatostatin receptor 3 (hSSTR3) and the multiple PDZ protein MUPP1.MUPP1 is a tight junction scaffold protein in epithelial cells, and as a result of the interaction withMUPP1 the hSSTR3 is targeted to tight junctions. Interaction with MUPP1 enables the receptor toregulate transepithelial permeability in a pertussis toxin sensitive manner, suggesting that hSSTR3can activate G-proteins locally at tight junctions.

Structured summary:MINT-6800756, MINT-6800770: MUPP1 (uniprotkb:O75970) and hSSTR3 (uniprotkb:P32745) colocalize(MI:0403) by fluorescence microscopy (MI:0416)MINT-6800587:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800562:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1 (uniprotkb:O75970) by two hybrid (MI:0018)MINT-6800622:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with PIST (uniprotkb: Q9HD26),Hsp70 (uniprotkb:P08107), Maguk p55 (uniprotkb: Q8N3R9), MAGI3 (uniprotkb:Q5TCQ9), ZO-2 (uni-protkb:Q9UDY2), ZO-1 (uniprotkb:Q07157) and MUPP1 (uniprotkb:O55164) by pull down (MI:0096)MINT-6800607, MINT-6801122:hSSTR3 (uniprotkb:P32745) physically interacts (MI:0218) with MUPP1(uniprotkb:O75970) by anti bait coimmunoprecipitation (MI:0006)

� 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

chemical Societies. Published by E

ctifying potassium channels;arby canine kidney; MUPP1,ge/ZO-1; PSD-95, postsynap-

sistance; TJs, tight junctions;, zonula occludens 1

(H.-J. Kreienkamp).dical School, One Joslin Place,

dez, 03202 Elche, Spain.ic Regulation, Barcelona, Spain.ipt.

Tight junctions (TJs) form the permeability barrier in epithelialtissues. A complex assembly of cell surface molecules such asoccludin and claudins with intracellular anchoring proteins includ-ing the PDZ domain proteins zonula occuldens 1 (ZO-1), ZO-2 andmultiple PDZ domain protein 1 (MUPP1) determines tightness andpermeability of TJs [1]. Various signaling molecules regulate for-mation and permeability of TJs, including heterotrimeric G-pro-teins [2,3]. Gai2, Gao and Ga12 subunits are localized at TJs orcan interact with the TJ scaffold ZO-1 [2–4]. However, so far ithas remained unclear which G-protein coupled receptors (GPCRs)

lsevier B.V. All rights reserved.

50 C.W. Liew et al. / FEBS Letters 583 (2009) 49–54

might be available to exert any influence on TJ function via theseG-proteins.

Here, we provide evidence for the regulation of transepithelialresistance by the G-protein coupled human somatostatin (SST)receptor 3 (hSSTR3). All SSTR subtypes (SSTR1-5) preferentially cou-ple to inhibitory, pertussis toxin (Ptx) sensitive G-proteins and exhi-bit common intracellular coupling patterns [5,6]. We show thathSSTR3 interacts with the tight junction protein MUPP1(multi-PDZ domain protein 1) via a PDZ domain of MUPP1. Thisinteraction allows for incorporation of receptors into TJs in a PDZ-dependent manner, and enables hSSTR3 to regulate transepithelialconductance.

2. Materials and methods

2.1. Materials

Rabbit anti-MUPP1 and guinea pig anti-hSSTR3 antisera weregenerated using GST-fusion proteins of MUPP1-PDZ10 and thehSSTR3 C-terminus, respectively, by custom immunization at Biog-enes GmbH, Berlin, Germany, and affinity-purified using immobi-lized antigens. Rabbit anti-SSTR3 has been described [7]. Anti-T7was from Novagen (Madison, WI); anti-b-catenin from BD Trans-duction Labs (Heidelberg, Germany).

cDNA coding for hSSTR3 was amplified from human genomicDNA and cloned into a modified pcDNA3 (Invitrogen, Leek, The

Fig. 1. Species specific interaction of the hSSTR3 C-terminus with MUPP1. (A) DomainOriginal positive ‘‘fish” clones derived from library screening (indicated by upper four linwith different bait plasmids to score for growth on plates lacking histidine (quadrupleseveral colonies after retransformation was seen as a positive (+) interaction, whereas tdifferences between individual MUPP1 constructs containing the region coding for PDZ10proteins were incubated with immobilized synthetic peptides derived from the C-terminprecipitates were analyzed by immunoblotting with MUPP1 antibody. (C) Coprecipitatiaffinity-purified, immobilized anti-MUPP, anti-shank (directed against the N-terminus of(In) and precipitates (P) were analyzed by Western blotting (WB) with the antibodies inlarger than the predicted size of 43 kDa, possibly due to glycosylation. In addition largemultimerized forms of the receptor. (D). HEK or MCF7 cellular lysates were subjected topeptides. Purified protein complexes were analyzed by SDS–PAGE; bands were identifie

Netherlands) providing an N-terminal T7-epitope tag (pcDNA3-NT7) in frame with the hSSTR3 coding sequence. Fusion constructswith mouse SSTR5 were generated by PCR and also cloned intopcDNA3-NT7.

2.2. Two-hybrid screening

A pGBKT7 (Clontech) derived bait plasmid containing a cDNAfragment coding for the C-terminal, intracellular domain of thehuman SSTR3 (Accession No. M96738; amino acids 317–419)was screened against a pretransformed human brain cDNA libraryin pACT-2 (Clontech). Plasmids from positive candidates wereisolated and sequenced.

2.3. Precipitation assays

Synthetic peptides corresponding to the C-termini of rat (KAS-TLSHL) and human (KSSTMRISYL) SSTR3 as well as the rat guany-late kinase associated protein/synapse associated proteinassociated protein (IYIPEAQTRL) were obtained from GenemedSynthesis Inc., San Francisco, CA. Peptides were immobilized onNHS-Sepharose (Amersham Pharmarcia Biotech) and used for pull-down assays as described [8]. Purified proteins were analyzed bySDS–PAGE, tryptic digestion and mass spectroscopy [8].

For immunoprecipitation, purified anti-MUPP1 antibody,anti-hSSTR3 or antibodies directed against a GST-fusion of the

structure of MUPP1, and result of two-hybrid screen with the hSSTR3 C-terminus.es), as well as clones generated for mapping the interaction, were combined in yeastdropout medium, lacking Trp, Leu, His and adenine). In each case robust growth ofhe lack of colonies was evaluated as a negative result (�); nd, not determined. Nowere observed. hSSTR3DCT lacks the C-terminal 8 residues. (B) Solubilized rat brain

i of GKAP (negative control, con), the rat (rS3) and the human SSTR3 (hS3). Input andon of hSSTR3 and MUPP1 from MCF7 cells. Cleared lysates were precipitated withhuman shank1; used here as negative control) and anti-hSSTR3, as indicated. Inputdicated. hSSTR3 migrates at a molecular weight of approximately 50 kDA, slightly

r bands are observed in the MUPP1 IP which may represent higher glycosylated oraffinity chromatography using immobilized hSSTR3 (hS3) or GKAP (con) C-terminald by mass spectrometry.

C.W. Liew et al. / FEBS Letters 583 (2009) 49–54 51

N-terminal 70 residues of human shank1 (as a non-related controlantibody) were bound to protein A/G sepharose (Santa Cruz Bio-technology, Santa Cruz, CA). After washing, antibodies were cross-linked to the beads with dimethylpimelimidate. MCF7 cells werelyzed in RIPA buffer, and immunoprecipitations were performedfrom cleared lysates using in each case 50 ll of antibody matrix.For subsequent detection of the hSSTR3, samples were denaturedin sample buffer at 60 �C for 10 min, to avoid aggregation.

2.4. Tissue culture

MCF7 cells (ATCC) were cultured in DMEM + 10% FCS, 10 mMHEPES, pH 7.4 and 10 lg/ml insulin. Keratinocytes were isolatedfrom human foreskin and cultured in KGM supplemented with0.07 mM Ca2+ (Cascade Biologics). For differentiation, they werecultured for 48 h in high Ca2+ (1.8 mM) medium with additives(0.2 lg/ml hydrocortisone, 5 lg/ml insulin and 50 lg/ml ascorbicacid). Madin-Darby canine kidney-II (MDCK-II) and HEK-293 cellswere cultured in DMEM + 10% FCS; in order to obtain polarizedepithelia, MDCK-II cells were grown on Transwell filters (Costar,Corning, NY). TER was measured using Millicell-ERS (Millipore,Eschborn, Germany). Statistical analysis was performed using Stu-dent’s t-test.

2.5. Immunocytochemistry

Cells growing on Transwell filters were fixed with 4% parafor-maldehyde in PBS and permeabilized with 0.1% Triton X-100 inPBS for 15 min at room temperature. After blocking (5% horse ser-

Fig. 2. The hSSTR3 C-terminus mediates targeting to cellular junctions in MDCK-II cellsanti-T7, green fluorescence; left) with the endogenous MUPP1 protein (affinity-purified aindicates colocalization. x and y Sections as well as x and z section obtained from the samtransmembrane region; PDZ-L, PDZ ligand motif. Black, hSSTR3; grey, mSSTR5; numbersof membrane preparations indicates equal T7-SSTR levels for cell lines expressing the trun4). The molecular weights of the two receptor variants are indicated. (D) MDCK-II cells ereceptor, left) and anti-b-catenin antibodies (middle). The merged picture is shown onkeratinocytes. (F) Colocalization of both proteins at cellular junctions using affinity-pur

um in PBS) cells were incubated with anti-T7 antibody (1:5000 inblocking solution) or purified polyclonal anti-MUPP PDZ10 anti-body (1:100) overnight at 4 �C, followed by 1 h of incubation withCy2-conjugated anti-mouse and Cy3-conjugated anti-rabbit sec-ondary antibody (1:500). Immunostaining was visualized by con-focal microscopy (Leica). Keratinocytes were grown on coverslipsto confluency, differentiated with high Ca2+-medium and incu-bated with primary antibodies (rabbit anti-MUPP 1:100, guineapig anti-SSTR3 1:20) followed by secondary antibody incubation(Cy3-anti-rabbit 1:400, AlexaFluor488-anti-guinea pig 1:1500).

3. Results

Using the C-terminus of the hSSTR3 as bait in a two-hybridscreen, we identified the PDZ domain protein MUPP1 (AccessionNo NM_003829; 16 independent clones; Fig. 1A) as potential inter-acting partner. Mapping experiments showed that interaction ismediated by the C-terminal PDZ ligand motif of the receptor, andPDZ domain 10 of MUPP1. Surprisingly, the rat SSTR3 which carriesa slightly modified PDZ ligand motif at its C-terminus, does notinteract with any of the PDZ domain proteins. The species-specificnature of the interaction was confirmed by peptide pulldownexperiments from rat brain. MUPP1 was readily detected in lysateand in precipitate obtained with human SSTR3 C-terminal peptide.Using the rat peptide for the pulldown, a faint band was detectedonly after overexposure of the blot (Fig. 1B). By homology model-ling [9] we identified a tyrosine at the �1 position of the humanpeptide ligand (histidine in the rat), as well as two additionalresidues present in the human SSTR3 C-terminus which determine

. (A and B) Costaining of T7-hSSTR variants expressed in MDCK-II cells (labelled bynti-MUPP1, red fluorescence, middle). Yellow staining in the merged images (right)e area are shown. The chimeric nature of the SSTR-constructs used is indicated; TM,refer to amino acid positions in the sequence. Bars, 10 lm. (C) Western blot analysiscated (lane 1) and the full-length fusion receptor (three different cell lines, lanes 2–xpressing the full-length fusion receptor were stained using anti-T7 (detecting the

the right. (E) Detection of hSSTR3 (left) and MUPP1 (right) in primary culturedified anti-hSSTR3 (left) and anti-MUPP1 (middle); Bars, 5 lm.

52 C.W. Liew et al. / FEBS Letters 583 (2009) 49–54

the high affinity of the human SSTR3 when compared to rSSTR3(Supplementary Fig. S1). A comparison with other SSTR3 sequencesshows that most vertebrate species actually carry a tyrosine at the-1 position (Table S1). Inability to bind PDZ10 of MUPP1 is there-fore a peculiarity of the rodent SSTR3, which however precludesthe use of mouse and rat model systems for functional studies.

Interaction of hSSTR3 with MUPP1 was demonstrated in a na-tive cellular system by coimmunoprecipitation of the endogenoushSSTR3 and MUPP1 proteins from lysates of human MCF-7 cells(Fig. 1C). We detected several MUPP1-specific bands in these cellsby Western blotting, consistent with the existence of several splicevariants (C.W.L., unpublished observations). Previous experiencehas shown that SSTRs are expressed at low levels, making itdifficult to detect receptor specific bands without prior enrichment[8]. Nevertheless, when precipitating MUPP1, we observedhSSTR3-specific bands in the precipitate at approximately 50 kDa(calculated: 46 kDa), as well as a smear of larger bands rangingfrom 70–90 kDa which presumably reflect differentially glycosyl-ated receptors. In the reverse experiment, MUPP1 was detectedin the precipitate when hSSTR3 was precipitated. As only a faintMUPP1-specific band could be observed, we assume that only afraction of total MUPP1 is associated with the hSSTR3.

Several attempts were made to define how the interaction withMUPP1 affects hSSTR3 function. In coexpression experiments inCHO cells, coupling of the hSSTR3 to its typical downstream effec-

Fig. 3. hSSTR3 regulates TER in MDCK-II cells in a biphasic manner. (A) Filters carryingindicated were subjected to Ca-switch; junctions were allowed to reform in the absence***Significantly different from control conditions, P < 0.001 as determined by Studentexpressing SSTR5S3C (C–D) were subjected to Ca-switch, and TER recovery in the absenceD were identical with the exception that different time points were included in D. The bi15 h after Ca-switch. Note that non-transfected cells do not respond to SST-treatment. **Pwas preincubated overnight at 400 ng/ml. Note that Ptx blocks the early reduction as wcells, values represent the mean ± S.E.M. determined from three independent experimenindependently generated cell lines.

tor, the G-protein gated inwardly rectifying potassium channel(GIRK; see Ref. [5]) was not affected by the interaction withMUPP1. In particular the activation and deactivation kinetics ofSST-14 induced currents were not altered in these experiments(Supplementary Fig. S2).

Next, we analyzed whether MUPP1 might integrate hSSTR3 intoa larger protein complex; taking advantage of the high affinity ofthe hSSTR3 C-terminus to MUPP1, we purified MUPP1-associatedproteins from human cell lines (Fig. 1D). Mass spectrometric anal-ysis indicated the presence of scaffold proteins ZO-1, ZO-2, MAGI-3, MAGUKp55/Pals, which are known components of epithelialtight junctions. Indeed, MUPP1 has been described as a scaffoldprotein in tight junctions in MDCK cells [10]. Interestingly, Ptx-sensitive G-proteins have also been localized at tight junctions[3,4]. However, so far no GPCR has been identified which couldactivate these G-proteins locally at the junctions. Therefore, weanalyzed a possible role of hSSTR3 in TJs of epithelial cells. Unfor-tunately, hSSTR3 overexpression causes apoptosis in many celllines [11] including the MDCK-derived lines tested (data notshown). However, we could generate MDCK-II derived cell linesexpressing fusion constructs of the N-terminal and transmem-brane part of mouse SSTR5 (which does not cause apoptosis) andthe C-terminus of hSSTR3. Analysis of transfected cells showed thatthe C-terminal PDZ ligand motif of the SSTR3 mediates targeting ofrecombinant receptors to tight junctions, as the SSTR5-derived

confluent layers of MDCK-II cells or MDCK-II cells expressing the SSTR-constructsor presence of agonist (1 lM SST-28). TER was measured 15 hours after Ca-switch.

’s t-test. (B–D) Time course. Non-transfected MDCK-II cells (B) or MDCK-II cellsor presence of 1 lM SST-28 was recorded at the times indicated. Conditions in C and

phasic nature of the reponse to SST is evident; the effect is reversed between 10 and< 0.001; *P < 0.05. (E) Sensitivity of SST induced changes to pertussis toxin (Ptx). Ptxell as the the late increase in TER induced by SST-28. For non-transfected MDCK-IIts. For transfected cells, values represent the mean ± S.E.M. determined from three

C.W. Liew et al. / FEBS Letters 583 (2009) 49–54 53

fusion receptor carrying the SSTR3 C-terminus (SSTR5S3C) colocal-ized with MUPP1 at the junctions, whereas a fusion receptor lack-ing the last 8 residues (SSTR5S3-DC) was distributed diffuselythroughout the cell surface (Fig. 2A and B). The expression levelof the receptor was not affected by the truncation, as determinedby Western blotting of membrane preparations derived fromtransfected cells (Fig. 2C). Costaining with an antibody againstb-catenin revealed that the chimeric receptor is indeed localizedapical relative to adherens junctions, further confirming targetingto tight junctions (Fig. 2D).

Recently, hSSTR3 was reported to be expressed in skin [12];therefore we analyzed the distribution of the endogenous hSSTR3in primary cultured keratinocytes which were differentiated byhigh Ca2+ concentrations; similar to MUPP1, the protein was foundhere also at cellular junctions, and colocalization of both proteinswas observed (Fig. 2E and F).

To analyze the functional role of the receptor at TJs, we em-ployed the so-called calcium switch paradigm; here Ca2+ is brieflyremoved to disrupt cellular junctions, leading to a complete loss oftransepithelial resistance. Reformation of tight junctions was theninduced by the addition of Ca2+, and the development of TER wasfollowed in the presence or absence of the SSTR agonist SST28.At 15 h after the switch, cells expressing the SSTR5S3C construct(which binds the MUPP1 PDZ domain) exhibited strongly increasedTER when agonist was present during recovery (Fig. 3A). Non-transfected cells or those expressing a construct which lacks theC-terminal PDZ motif (SSTR5S3CDC) on the other hand did not re-spond to SST-treatment. Analysis of TER at earlier times after Ca-switching revealed that the response to SST in MDCK cells is bipha-sic, as TER values actually declined in the SSTR expressing cell linerelative to control cells (non-treated, or treated but not transfec-ted) during the first 10 h after the Ca-switch (Fig. 3B–E), but in-creased strongly at later time points (15–23 h). After that, TERgradually decreased again. Importantly, both the initial decreaseand the subsequent increase in TER values induced by SST28 weresensitive to pertussis toxin treatment (Fig. 3E). This indicates thattargeting of hSSTR3 to tight junctions via its C-terminal interactionenables localized control of TJ permeability via heterotrimeric G-protein dependent signalling.

4. Discussion

Our data indicate a strong and specific interaction between thehuman somatostatin receptor subtype 3 and PDZ domain 10 ofMUPP1. We have previously established that the C-termini ofsomatostatin receptors may interact with PDZ domain proteins[8]; in fact, all SSTR subtypes contain potential PDZ ligand motifs.PDZ-type interactions have previously been shown not to affectthe ‘‘normal” signal transduction pathways, but rather to couplea given receptor to additional, new downstream signalling events[13]. This is also evident here, as the interaction with MUPP1 didnot have any influence on coupling of hSSTR3 to the GIRK1/2, awell established effector of SSTRs [5]. In epithelial cells, targetingof the hSSTR3 to tight junctions depends on its PDZ ligand motif,implying MUPP1 to be responsible for the junctional localization.Receptor lacking the PDZ ligand is diffusely distributed throughoutcells, whereas only the receptor with the ligand motif is foundcolocalized with MUPP1 at tight junctions. Currently, our data sug-gest that due to its interaction with a MUPP1-based complex at TJs,the receptor can control the formation and permeability of epithe-lial barriers, as indicated by TER measurements in transfectedMDCK-II cells. Further work will show whether other MUPP1-asso-ciated GPCRs such as serotonin 5-HT2 receptors or the melatonin 1receptor [14–16] might contribute to the regulation of TJs. Interest-ingly, interaction of the 5-HT2C receptor with MUPP1 is inhibited

by the agonist-induced phosphorylation of the receptor within itsC-terminal PDZ ligand motif [17], suggesting that targeting ofreceptors to cellular junctions may be dynamically regulated uponreceptor activation. We did not analyze this aspect with respect tothe hSSTR3/MUPP1 interaction. However, our previous analysis ofthe rat SSTR3 indicated that agonist-dependent phosphorylationsites in the C-terminal domain are not located within the putativePDZ ligand motif, but more proximal to the plasma membrane[18]. Most of these sites are conserved from rat to human SSTR3.Subsequent to phosphorylation, receptors are internalized [18]; itwill be important to analyze whether the interaction with MUPP1is maintained during endocytosis, and whether such a redistribu-tion of MUPP1 contributes to the reduction of TER observed inthe early phase of agonist treatment.

Taken together, the expression of the SSTR3 in epithelial tissues,such as prostate [19] and kidney [20] and skin (see above), and theability of the receptor to regulate tight junctions indicate that SSTcan affect permeability and biogenesis of these biological barriers.

Acknowledgements

We thank Hans-Hinrich Hönck and Sönke Harder for excellentassistance; Kai Simons (MPI Dresden) for MDCK-II cells; ShoichiroTsukita (Kyoto, Japan) for anti-MUPP1 antibody. Financial supportfrom DFG (Ri 192/24-1; SFB545/B7 to D.R. and H.-J.K.) and the EC(QLG3-CT-1999-00908 to D.R.) is gratefully acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.febslet.2008.11.048.

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