-arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism

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   -arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism
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   Article b -arrestin- 1  mediates the TCR-triggered re-routingof distal receptors to the immunological synapse bya PKC-mediated mechanism Elena Fern  andez-Arenas 1 , Enrique Calleja 1 , Nadia Mart  ı nez-Mart  ı n 1 , Severine I Gharbi 2 ,Rosana Navajas 2 , Noel Garc  ı a-Medel 1 , Petronila Penela 1 , 3 , Antonio Alcam  ı 1 , Federico Mayor Jr  1 , 3 , Juan P Albar  2 & Balbino Alarc  on 1 ,* Abstract T-cell receptors (TCR) recognize their antigen ligand at the inter-face between T cells and antigen-presenting cells, known as theimmunological synapse (IS). The IS provides a means of sustainingthe TCR signal which requires the continual supply of new TCRs.These are endocytosed and redirected from distal membrane loca-tions to the IS. In our search for novel cytoplasmic effectors, wehave identified  b -arrestin- 1  as a ligand of non-phosphorylatedresting TCRs. Using dominant-negative and knockdown approacheswe demonstrate that  b -arrestin- 1  is required for the internali-zation and downregulation of non-engaged bystander TCRs.Furthermore, TCR triggering provokes the  b -arrestin- 1 -mediateddownregulation of the G-protein coupled chemokine receptor CXCR 4 , but not of other control receptors. We demonstrate that b -arrestin- 1  recruitment to the TCR, and bystander TCR andCXCR 4  downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of   b -arrestin- 1  at Ser  163 .This mechanism allows the first triggered TCRs to deliver a stopmigration signal, and to promote the internalization of distal TCRsand CXCR 4  and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal. Keywords  arrestin; chemokine receptors; GPCR; PKC; receptor crosstalk; signalsustainment Subject Categories  Signal Transduction; Immunology; Membrane & Intra-cellular Transport DOI  10 . 1002 /embj. 201386022  | Received  18  June  2013  | Revised  18  November  2013  | Accepted  9  December   2013  | Published online  6  February  2014 EMBO Journal (  2014) 33, 559  –  577 See also:  VV Gurevich  &  EV Gurevich  (March  2014 ) Introduction Cells integrate extracellular cues that activate different membranereceptors, which in turn modulate the activity of a panel of transcriptionfactors, and/or mediate the rearrangement of the cytoskeleton andorganelles. This signal integration does not always occur in thenucleus or through the cytoplasmic scaffold of signaling proteins.Moreover, some membrane receptors crosscommunicate, wherebyone receptor type uses the signaling machinery of another receptor(Natarajan & Berk, 2006). A typical example of this is the crosstalkbetween the 7-transmembrane G protein-coupled receptors (GPCRs)and receptor tyrosine kinases (RTKs) initiated upon binding of eitherGPCR or RTK ligands (Natarajan & Berk, 2006; Shenoy & Lefkowitz,2011). As such, ligand binding to RTKs can result in the appropria-tion of several components of the GPCR-mediated signaling machin-ery, including  b -arrestin and G protein-receptor kinases (Shenoy & Lefkowitz, 2011). This crosstalk may be mediated by RTK-triggeredactivation of GPCR-associated G proteins, or even through directinteractions (Piiper & Zeuzem, 2004; Delcourt  et al , 2007).The TCR interacts functionally and physically with the CXCR4chemokine receptor, a GPCR involved in the migration of T cells intolymphoid organs in response to gradients of the chemokine CXCL12(Peacock & Jirik, 1999; Molon  et al , 2005; Kumar  et al , 2006). TCRtriggering provokes the desensitization of T cells to CXCL12, block-ing their migratory behavior and facilitating the formation of a stablecontact between T cells and antigen presenting cells (APCs) (Brom-ley  et al , 2000; Molon  et al , 2005). Upon contacting an antigen-loaded APC, T cells rearrange their actin cytoskeleton and form atight apposition with the APC membrane, generating a structureknown as the immunological synapse (IS) (Dustin  et al , 2010; Yoko-suka & Saito, 2010). The TCR binds to the peptide-major histocom-patibility complex (pMHC) ligand at the periphery of the IS, formingmicron-sized clusters termed microclusters. TCRs transmit signalsfrom within these microclusters, which move centripetally to form a 1  Centro de Biolog  ı a Molecular   “ Severo Ochoa ” , Consejo Superior de Investigaciones Cient  ı ficas and Universidad Aut  onoma de Madrid, Madrid, Spain 2  Proteomics Facility, Centro Nacional de Biotecnolog  ı a/Consejo Superior de Investigaciones Cient  ı ficas (CNB/CSIC), Madrid, Spain 3  Instituto de Investigaci  on Sanitaria La Princesa, Madrid, Spain*Corresponding author. Tel: + 34 911964555 ; Fax: + 34 911964420 ; E-mail: balarcon@cbm.uam.es ª 2014  The Authors  The EMBO Journal  Vol  33  | No  6  |  2014  559 Published online: February 6, 2014  central aggregation of TCRs, which results in the termination of TCRsignaling and their subsequent removal from the center of the IS by aphagocytotic-like mechanism (Alarcon  et al , 2011; Martinez-Martin et al , 2011). For full activation, exhausted TCRs must be replaced bynew TCRs to engage pMHC, thereby sustaining TCR signaling forhours (Huppa  et al , 2003). Thus, while horizontal movements of TCRs initially positioned at the T cell-APC contact site can mediateinitial signaling events, distal TCRs must be internalized and rerout-ed to the IS in order to sustain the TCR signal (Das  et al , 2004).At least two independent mechanisms can be used to internalizepMHC-triggered TCRs: a clathrin-coated pit mechanism and a phagocytic-like mechanism. However, in addition to inducing the internal-ization of pMHC-engaged TCRs, TCR triggering provokes the inter-nalization and downregulation of non-contacted bystander TCRs(Niedergang  et al , 1998; San Jose  et al , 2000; Monjas  et al , 2004).This co-modulation of bystander TCRs occurs via a clathrin-coatedpit-dependent mechanism that requires the activation and participa-tion of protein kinase C (PKC) isoforms (Monjas  et al , 2004; vonEssen  et al , 2006).TCR engagement by pMHC induces the tyrosine phosphorylationof the so-called immunoreceptor tyrosine-based activation motifs(ITAMs), which consist of a double stretch of tyrosine and leucine(or isoleucine) residues with the consensus sequence YxxL/I(x)6-9YxxL/I. The dual phosphorylated ITAMs serve as high-affinitydocking sites for signaling proteins containing tandem SH2 domains,such as the Syk-family tyrosine kinase ZAP-70. However, in theirnon-phosphorylated form, ITAMs are docking sites for other pro-teins such as adaptins, which can recognize each of the YxxL/Imotifs and participate in endocytosis (Szymczak & Vignali, 2005).Other proteins that bind non-phosphorylated ITAMs include TC21(also known as RRas2), a member of the Ras-related subfamily of GTPases, and EB1, a plus-end microtubule-tracking protein (Delgado et al , 2009; Martin-Cofreces  et al , 2012).To identify novel TCR effectors that bind to non-phosphorylatedITAMs, we employed a proteomic approach using synthetic peptidesas baits. Among others, we identified  b -arrestin-1 ( b -Arr1; alsoknown as arrestin-2) as a protein that binds to non-phosphorylatedITAMs in a manner dependent on TCR triggering and mediated byPKC.  b -Arr1 binding induces the co-modulation of distal bystanderTCRs and their re-routing to the IS, accompanied by CXCR4. Wedemonstrate that this mechanism is required to supply the IS withnew TCRs and hence, to sustain the TCR signal. Results b -arrestin- 1 is a direct cytoplasmic ligand of the TCR To characterize new ligands of TCR ITAMs, we employed a proteo-mic approach using a biotinylated synthetic peptide correspondingto the first ITAM (membrane-proximal, CD3 f a; Fig 1A) of CD3 f  in itsunphosphorylated form as bait. For comparison, dual tyrosine phos-phorylated peptides corresponding to the first and third ITAMs(CD3 f aP and CD3 f cP) were run in parallel. The three biotinylatedpeptides were incubated with post-nuclear detergent lysates of thehuman T cell leukemia line Jurkat, which were incubated in theabsence or presence of anti-CD3 to trigger TCRs. Proteins in the celllysate that co-purified with the three peptides after incubation withstreptavidin-agarose beads were characterized by tandem mass spec-trometry (MS/MS). In the ITAM-bound fractions we identified 105proteins from unstimulated cell lysates and 176 proteins from lysatesof TCR-triggered cells (Supplementary Fig S1A and Table S1). Someof these ITAM-interacting proteins have been identified previouslyas phosphorylated substrates downstream of the TCR (Mayya  et al ,2009). Proteins involved in intracellular/vesicular transport, endocy-tosis, cytoskeletal function and signal transduction were particularlyimportant for TCR function (Supplementary Fig S1B and Table S1).We selected nine proteins for validation by immunoblotting afterpull-down with biotinylated peptides. Four protein interactions werevalidated (Fig 1B). These proteins are known to participate in endo-cytosis (McDonald  et al , 1999; McMahon & Boucrot, 2011; Meyer et al , 2012):  b -Arr1, vesicle-fusing ATPase (NSF), valosin containingprotein (VCP) and clathrin heavy chain (CHC). Interestingly, the fourvalidated proteins only interacted with CD3 f  ITAMs in T cells thathad been previously stimulated with anti-CD3, suggesting that theirbinding to the TCR is regulated by TCR signaling. However, whileCHC and VCP showed no preference for tyrosine phosphorylated(CD3 f aP and CD3 f cP) versus unphosphorylated (CD3 f a) ITAMs,NSF and  b -Arr1 appeared to bind preferentially the unphosphory-lated ITAM (Fig 1B).While the function of   b -Arr1   in GPCRs has been well described(Shukla  et al , 2011), its role as a TCR effector is unknown. Thus,we investigated the possible function of the interaction between b -Arr1 and the TCR.  b -Arr1 was recruited to the TCR of T cells in aTCR triggering-dependent manner (Fig 1C), indicating that the b -Arr1-ITAM interaction (Fig 1B) is not an artifact of peptide over-expression. Using recombinant purified  b -Arr1 we found that thisprotein interacted directly with two unphosphorylated ITAMs(CD3 f a and CD3 f b) and less strongly with two phosphorylatedITAMS (Fig 1D). These results suggest that  b -Arr1 is a direct bindingpartner of the TCR. To characterize the region of   b -Arr1 involved inthis interaction, we used 2 constructs that express either the first 382amino acids ( b -Arr1 [1-382])(Key  et al , 2005) or the last 99 aminoacids ( b -Arr1 [319-418])(Braun  et al , 2003) of this protein (Fig 2A).The 319-418 fragment contains binding sites for CHC and for theadaptin complex AP2 (Fig 2A), and it therefore plays an importantrole in  b -Arr1-mediated GPCR endocytosis (Krupnick  et al , 1997;Kern  et al , 2009). Both fragments were expressed as GFP fusion pro-teins in HEK-293T cells together with (His) 6 -tagged CD3 f . Both trun-cated proteins co-purified with (His) 6 -tagged CD3 f  to a similar extentas to the full length wild-type (WT)  b -Arr1 ([1-418], Fig 2B). Thetwo truncated proteins share amino acids 319-382 (Fig 2A), indicat-ing that this sequence must mediate the association of   b -Arr1 withITAMs. Indeed, a GST fusion protein with this sequence co-purifiedwith the unphosphorylated ITAMs, confirming that the 319-382sequence contains the ITAM-binding site (Fig 2C). Notably, despitesharing 76% amino acid identity,  b -Arr1 but not  b -Arr2 (also knownas arrestin-3) interacted with CD3 f a (Fig 1B), suggesting that anon-conserved sequence within amino acids 319-382 mediates thisinteraction. A comparison of mammalian protein sequences showsdifferent stretches of amino acids that are conserved in  b -Arr1 butnot in  b -Arr2 (Supplementary Fig S2), suggesting that one or severalof these sequences could be responsible for the interaction withITAMs. The recruitment of WT  b -Arr1 (1-418) and the truncated b -Arr1 (1-382) and  b -Arr1   (319-418) fragments to the TCR wasstudied in transiently transfected Jurkat T cells stimulated with The EMBO Journal  Receptor re-routing mediated by  b -arrestin- 1  Elena Fern   andez-Arenas et al 560  The EMBO Journal  Vol  33  | No  6  |  2014  ª 2014  The Authors Published online: February 6, 2014  superantigen-loaded APCs. Whereas binding to the TCR by the WTand the  b -Arr1 (1-382) constructs was clearly induced by TCRtriggering,  b -Arr1 (319-418) bound strongly even in basal conditions(Fig 2D), suggesting that the ITAM-binding motif is constitutivelyexposed in the  b -Arr1 (319-418) fragment. These results suggestimportant differences in the manner in which  b -Arr1 binds to TCRsand GPCRs, as the  b -Arr1 (319-418) construct alone does not bind toGPCRs (Krupnick  et al , 1997). b -arrestin- 1 mediates the downregulation of bystander TCRs Given that the  b -Arr1 (319-418) contains CHC and AP2 bindingsites, and that it binds constitutively to the TCR, we investigatedthe effect of over-expressing this construct on TCR plasmamembrane expression. TCR expression was constitutively reduced2  –  3 fold in Jurkat cells transfected with the  b -Arr1 (319-418) con-struct as compared with control cells (pEGFP vector) or cellstransfected with the WT or  b -Arr1 (1-382) constructs (Fig 3A),suggesting that the  b -Arr1 (319-418) construct downregulates TCRexpression in resting conditions. Interestingly however, over-expression of   b -Arr1 (319-418), and to a lesser extent  b -Arr1 (1-382), delayed ligand (superantigen)-induced downregulation of theTCR (Fig 3B), an inhibitory effect that was particularly notableduring the first 20 min of stimulation. Of note, cells transfectedwith  b -Arr1 (1-382) had basal TCR levels similar to those trans-fected with the WT construct, and therefore the inhibition of  30505018010075 Load  ζ a - + - + - + + ζ aP ζ cP β -Arr1- β -Arr2-NSFVCPCHCStim : pull-down βαζζδ ε a abbc c   PO 4 PO 4 PO 4 PO 4 PO 4 PO 4 PO 4 PO 4 PO 4 PO 4 PO 4 PO 4 aP ζ cP ζγ ε   GQNQLYNELNLGRREEYDVLDKRRBiotinAhx CD3 ζ a GHDGLY( PO 4 H 2 )QGLSTATKDTY( PO 4 H 2 )DALHMQA CD3 ζ cP Biotin AhxGQNQLY( PO 4 H 2 )NELNLGRREEY( PO 4 H 2 )DVLDKRR CD3 ζ aP Biotin Ahx AB CD co-IP - + Stim:IP: anti-CD3WB: CD3 ζ Cell lysateWB: β -Arr1    R  e   l  a   t   i  v  e   I   T   A   M   /         β   -   A  r  r   1   i  n   t  e  r  a  c   t   i  o  n 6428    M  o  c   k    ζ    a    ζ    a   P    ζ    c   P    ζ     b P   = 0.0009 P   = 0.026 0Pd:ITAMsWB: β -Arr1WB: β -Arrb1LoadWB: CD3 ζ LoadITAMS    M  o  c   k    ζ    a    ζ    a   P    ζ    c   P    ζ     b pull-down kDakDa 50 kDa 5013 Figure  1 . Identification of   b -Arr  1 as an ITAM-binding protein. A Cartoon of a TCR and the sequence of the ITAM-encoding biotin-labelled synthetic peptides used in this paper.B Validation of a set of ITAM-interacting proteins in a pull-down and immunoblot assay. ITAM-interacting proteins from lysates of control (   ) and anti-CD 3-stimulated(+) Jurkat T cells were pulled-down with the indicated biotinylated ITAMs and identified in Western blots (WB) probed with the specific antibodies indicated. Todemonstrate the reactivity of the antibodies, 5% of the pull-down input was analyzed directly.  b -Arr1,  b -arrestin 1;  b -Arr2,  b -arrestin 2; NSF, vesicle-fusing ATPase;VCP, valosin containing protein; CHC, clathrin heavy chain (representative of three experiments). C T-cell stimulation induces the recruitment of   b -Arr  1 to the TCR. Murine T-cell lymphoblasts were stimulated with anti-CD3 for 20 min.  b -Arr1 co-purified with theTCR in anti-CD3 immunoprecipitates. Whole cell lysates were used as controls of expression (representative of three experiments). D  b -Arr  1 interacts directly with the unphosphorylated  f a and  f b ITAMs. Recombinant  b -Arr1 was incubated with an irrelevant biotinylated peptide (mock) or with theindicated biotinylated ITAMs and pulled-down with streptavidin-sepharose beads. The initial input of recombinant  b -Arr1 and biotinylated peptides was estimatedby immunoblotting with anti- b -Arr1 and anti-CD3 f  antibodies. The densitometric intensity of the co-purified  b -Arr1 bands was compared to that of the initial inputand all values are expressed relative to the control condition (mock), considered as 1. The data represent the mean  s.d. of three independent experiments. Source data are available online for this figure. Elena Fern   andez-Arenas et al  Receptor re-routing mediated by  b -arrestin- 1  The EMBO Journal 561 ª 2014  The Authors  The EMBO Journal  Vol  33  | No  6  |  2014 Published online: February 6, 2014  ligand-triggered TCR downregulation could not be attributed toreduced initial TCR expression.There was significant co-localization of internal TCR and CHC in Jurkat T cells expressing either WT  b -Arr1 or  b -Arr1 (319-418) whenincubated for 5 min in the presence (Fig 3C, left panel) or absenceof anti-CD3 (Supplementary Fig S3). However, the co-localizationcoefficient between the transfected  b -Arr1 constructs and TCR orCHC was much higher for  b -Arr1 (319-418) (Fig 3C, middle paneland plot). Hence,  b -Arr1 (319-418) over-expression would appear toinduce the accumulation of the TCR in CHC-positive endocytoticvesicles, thereby accounting for the observed reduction in TCRsurface expression (Fig 3A and B). The effect of this construct in A N-domain(1-185)C-domain(186-418) 412CHCAP2SS β -Arr1 (1-418) β -Arr1 (1-382) β -Arr1 (319-418) β -  Arr1 (319-382) 41841838238231931911 B    R  e   l  a   t   i  v  e   I   T   A   M   /        β   -   A  r  r   1   i  n   t  e  r  a  c   t   i  o  n    M  o  c   k    ζ    a  ζ     b 12345 P   = 0.004 P   = 0.00 0 C GST Pd:ITAMs WB: GSTWB: CD3 ζ Load WB: GST ITAMs    M  o  c   k   M  o  c   k β -Arr1 Pd:ITAMs GST  GST- β - Arr1 (319-382) ζ a ζ b GST β -Arr1 ζ a ζ b    R  e   l  a   t   i  v  e        β   -   A  r  r   1   /   C   D   3        ζ    b   i  n   d   i  n  g 0.20.40.6   β  -  A  r  r  1   (   1 - 4  1  8  ) 0   β  -  A  r  r  1   (   3  1  9 - 4  1  8  )   β  -  A  r  r  1   (   1 -  3  8  2  ) β -Arr1 (1-418) WB: CD3 ζ WB: GFP    P   d  :   N   i  -   N   T   A   P   d  :   N   i  -   N   T   A   β  -  A  r  r  1   (   1 - 4  1  8  )   β  -  A  r  r  1   (   3  1  9 - 4  1  8  )   β  -  A  r  r  1   (   1 -  3  8  2  )   β -Arr1 (1-382) β -Arr1 (319-418)   c  e   l   l   l  y  s  a   t  e WB: GFP   E  m  p  t  y   v e c  t o  r   β  -  A  r  r  1   (   1 - 4  1  8  )   β  -  A  r  r  1   (   3  1  9 - 4  1  8  )   β  -  A  r  r  1   (   1 -  3  8  2  )  E  m  p  t  y   v e c  t o  r pull-down pull-down 75503725755037253725372530 kDa kDa kDa 13 D    C   D   3        ζ       /       β   -   A  r  r   1   b   i  n   d   i  n  g 0.41.201020300 Time (min) β -Arr1 (1-418) β -Arr1 (319-418) β -Arr1 (1-382)0.8 β -Arr1 (1-418)  β -Arr1 (319-418) β -Arr1 (1-382) 0 10 20 30 0 10 20 30 0 10 20 30 WB: CD3 ζ CD3 ζ WB: GFP SEE Superantigen stimulation Stim(min): Ip:GFP Ip:GFP β -Arr1 (1-418) β -Arr1 (1-382) β -Arr1 (319-418) co-IP 753730 kDa The EMBO Journal  Receptor re-routing mediated by  b -arrestin- 1  Elena Fern   andez-Arenas et al 562  The EMBO Journal  Vol  33  | No  6  |  2014  ª 2014  The Authors Published online: February 6, 2014  promoting constitutive TCR internalization while inhibiting ligand-induced TCR down-regulation was also evident when the inside/outside ratio of TCR fluorescence intensity was evaluated by confo-cal microscopy. In cells transfected with WT  b -Arr1, increasedinside/outside ratios were observed 5 and 10 min after stimulation.By contrast, the inside/outside ratio was higher at time 0 in cellstransfected with the  b -Arr1 (319-418) construct than in cells trans-fected with the WT construct, although it did not increase furtherupon stimulation (Fig 3C, right panel). These results suggest that b -Arr1 (319-418) promotes the internalization and sequestering of TCRs in endocytotic vesicles, and blocks any increase in TCR down-regulation provoked by the ligand.The over-expression of the  b -Arr1 (319-418) construct in Jurkatcells suggests that  b -Arr1 mediates the ligand-induced down-regula-tion of TCRs. To corroborate this finding using an alternativemethod, we used a panel of 3 shRNA constructs to silence  b -Arr1mRNA expression. When transfected into the 2B4 murine T cellhybridoma, the 3 constructs produced a variable reduction in  b -Arr1mRNA expression: transfection of shRNA construct 1 reduced b -Arr1 mRNA expression by up to 80% of that with a non-targetshRNA control construct (Fig 3D, left panel);. shRNA construct 3was slightly less effective than construct 1; while construct 2 had nosignificant effect on mRNA expression. The effect of these 3 shRNAconstructs on  b -Arr1 mRNA expression correlated with their effecton antigen-induced TCR down-regulation in 2B4 cells (Fig 3D, mid-dle panel). Thus, shRNA construct 1 was used to knockdown  b -Arr1expression in all subsequent experiments. It induced a decrease in b -Arr1 protein expression of over 50% in 2B4 cells (Fig 3D, rightpanel). Unlike expression of the  b -Arr1 (319-418) construct, theknockdown approach did not affect the levels of basal TCR expres-sion (data not shown).We and others have previously reported that TCR triggeringinduces the down-regulation of non-triggered bystander TCRs via aTCR signaling-dependent mechanism (Niedergang  et al , 1998; San Jose  et al , 2000; Bonefeld  et al , 2003; Geisler, 2004; Monjas  et al ,2004). To determine whether  b -Arr1 mediates this trans-actingeffect, we used CD8 + T cells from double transgenic mice express-ing two TCRs with different antigen specificities: the OT-I TCR andthe HY TCR (Fig 4A). While the OT-I TCR responds to OVAp, theHY TCR responds to an antigen (SMCY) expressed only in malecells. We studied in CD8 + T cells from double transgenic femalemice (Fig 4B) if stimulation of 1 of the TCRs (e.g., OT-I)down-regulated the engaged OT-I TCR complexes and induced theco-modulation of non-engaged HY and OT-I TCR complexes(Fig 4A). As expected, stimulation with OVAp induced the down-regulation of both TCRs (Fig 4C). Of note, while some of the down-regulated OT-I TCRs may have been directly engaged by ligand, allthe down-regulated HY TCRs were bystander. Down-regulation of bystander HY TCRs was not due to unforeseen cross-reactivity withOVAp as stimulation of CD8 + T cells from single HY TCR transgenicmice with OVAp did not induce the down-regulation of HY TCRs(Fig 4D). By contrast, stimulation of single HY TCR transgenic orOT-I transgenic T cells with their cognate antigens (SMCYp andOVAp, respectively) resulted in HY and OT-I TCR down-regulation,respectively (Fig 4D).The role of   b -Arr1 in the down-regulation of bystander TCRs wasassessed in double transgenic CD8 + T cells transduced with theshRNA construct 1 or a control construct. This shRNA reduced b -Arr1 protein expression by  ~  50%, compared with a control scram-bled shRNA construct, but it did not affect  b -Arr2 protein expression(Fig 4E).  b -Arr1 knockdown inhibited the OVAp-triggered down-regulation of the stimulated (OT-I), but also of the non-stimulated (HY)TCRs (Fig 4F), suggesting that  b -Arr1 mediates the down-regulationin  trans  of unbound TCRs. We next investigated the mechanismunderlying  b -Arr1 recruitment to non-engaged receptors in doubleTCR transgenic T cells using specific inhibitors. As expected (San Jose  et al , 2000; Monjas  et al , 2004), PP2 inhibition of Src kinases,immediate transducers of TCR signals, blocked the down-regulationof bystander HY TCRs (Fig 4G), indicating that activation of TCR-triggered signaling is required to co-modulate non-engaged TCRs.Moreover, the PKC inhibitor G € o6976 (Martiny-Baron  et al , 1993)also blocked down-regulation of bystander TCRs (Fig 4G), furtherdemonstrating that this effect is mediated by PKC, as reported previ-ously (von Essen  et al , 2006). TCR-triggering induces the phosphorylation of   b -arrestin- 1 atSer163 by PKC, required for TCR binding We hypothesized that regulation of   b -Arr1 recruitment to the TCRoccurs through the phosphorylation of   b -Arr1 by PKC. We transfected Jurkat T cells with the WT  b -Arr1 (1-418)-GFP construct andstimulated them with superantigen. Phosphorylation of transfected b -Arr1 by PKC was assessed in anti-GFP immunoprecipitates byimmunoblotting with an anti-phosphoserine antibody specific Figure  2 .  b -Arr  1 is a direct cytoplasmic ligand of the TCR. A Schematic representation of   b -Arr  1 showing the regions of   b -Arr1 interacting with various partners and the various deletion mutants. CHC, clathrin heavy chain,AP2, adaptor protein complex 2. B CD 3 f  interacts with both the  b -Arr1 (1-382) and  b -Arr1 (319-418) constructs. HEK-293T cells were co-transfected with His 6 -tagged CD3 f  and WT  b -Arr1 (1-418) (fulllength protein), or either of the 2 truncated mutants [ b -Arr1 (1-382) and  b -Arr1 (319-418)], all fused to GFP, or with empty vector (expressing non-fused GFP).Ni-NTA beads were used to pull-down CD3 f  and co-purify the  b -Arr1 constructs. Data were normalized to the total expression of GFP-fused proteins and expressedas the mean  s.d. of three independent experiments. C Amino acids  319-382 of   b -Arr1 mediate its binding to the ITAMS. Unphosphorylated biotinylated ITAMS (  f a and  f b) were incubated with purified GST or purifiedGST- b -Arr1 (318-382) fusion protein before pull-down with streptavidin-sepharose beads. Co-purified proteins were detected after immunoblotting with anti-GST.Data were normalized to the total expression of GST proteins and expressed as the mean  s.d. of two independent experiments. D High basal levels of   b -Arr  1 (319-418) mutant binding to the TCR in non-stimulated cells. Jurkat cells were transiently transfected with vectors encoding theindicated GFP fusion proteins and stimulated with unloaded (0 min) or SEE superantigen-loaded Raji APCs for the times indicated (10, 20 or 30 min).Immunoprecipitation was carried out using an anti-GFP antibody and the co-purified TCR was detected in Western blots with anti-CD3 f . Data were normalized tothe total expression of the GFP-fusion proteins. Results shown are representative of three independent experiments. Source data are available online for this figure. ◂ Elena Fern   andez-Arenas et al  Receptor re-routing mediated by  b -arrestin- 1  The EMBO Journal 563 ª 2014  The Authors  The EMBO Journal  Vol  33  | No  6  |  2014 Published online: February 6, 2014
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