Dopamine receptor D3 regulates endocytic sortingby a Prazosin-sensitive interaction with thecoatomer COPIXin Zhanga,b,c, Wenchao Wanga,c, Anne V. Bedigiana,b, Margaret L. Coughlind, Timothy J. Mitchisond,and Ulrike S. Eggerta,b,e,1

aDana-Farber Cancer Institute, bDepartment of Biological Chemistry and Molecular Pharmacology, and dDepartment of Systems Biology, Harvard MedicalSchool, Boston, MA 02115; cHigh Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, 230031, People’s Republic of China; and eDepartment ofChemistry and Randall Division of Cell and Molecular Biophysics, King’s College London, London SE1 1UL, United Kingdom

Edited* by Christopher T. Walsh, Harvard Medical School, Boston, MA, and approved June 19, 2012 (received for review May 8, 2012)

Macromolecules enter cells byendocytosis andare sorted todifferentcellular destinations in early/sorting endosomes. Themechanismandregulation of sorting are poorly understood, although transitionsbetween vesicular and tubular endosomes are important. We foundthat the antihypertensive drug Prazosin inhibits endocytic sorting byan off-target perturbation of the G protein-coupled receptor dopa-mine receptor D3 (DRD3). Prazosin is also a potent cytokinesis inhib-itor, likely as a consequence of its effects on endosomes. Prazosinstabilizes a normally transient interaction between DRD3 and thecoatomer COPI, a complex involved in membrane transport, andshifts endosomal morphology entirely to tubules, disrupting cargosorting. RNAi depletion ofDRD3 alone also inhibits endocytic sorting,indicating a noncanonical role for a G protein-coupled receptor.Prazosin is a powerful tool for rapid and reversible perturbationof endocytic dynamics.

small-molecule inhibitor | endocytic tubulation |unconventional G protein-coupled receptor function |drug off-target effects | membrane trafficking

Gprotein-coupled receptors (GPCRs) are the most importantchemosensing receptors in animals and the targets of many

therapeutic drugs. They have mostly been studied from theperspective of signal transduction and pharmacology, but therehave been hints that GPCRs are important regulators of basiccellular processes (1, 2). The dopamine receptors (D1–5) areGPCRs with important functions in the brain, and they are tar-geted by numerous drugs used to treat neurological disordersranging from Parkinson disease to schizophrenia. Although thereis a large amount of literature about the regulation of GPCRsignaling by endocytosis, much less is known about how or ifGPCRs, in turn, regulate endocytosis (3–5). Our data show thatthe dopamine receptor D3 (DRD3) plays an unexpected role inendocytic sorting.Many different proteins and complexes enter cells by endo-

cytosis, and they must be rapidly sorted for transport to differentlocations in the cell. For example, some cargoes are recycled tothe plasma membrane, whereas others are sent to lysosomes.Sorting occurs in specialized compartments called early or sort-ing endosomes. Highly dynamic trafficking occurs between thesecompartments and multiple other cellular compartments, drivenby sorting, budding, fission, and fusion reactions (6). Althoughsome individual steps in endocytic sorting have been elucidated,their coordination in cells remains mysterious. Dynamic tran-sitions between vesicular and tubular endosomes seem to be keyfactors in determining the fate of endocytic cargoes; the coat-omer complex COPI may play a role in these dynamics (7, 8), butprecisely how these transitions are regulated is unclear. Onereason that it has been difficult to elucidate sorting endosomedynamics in living cells has been the lack of small-molecule toolsto rapidly and reversibly perturb them. Elucidation of Golgidynamics benefitted greatly from use of Brefeldin (9), a smallmolecule that inhibits Arf guanine-nucleotide exchange factor(ArfGEF), perturbing the functions of COPI at the Golgi (10).

Prazosin, which we describe here as a tool for endocytosisresearch, is an important drug that has been used clinically fordecades to treat hypertension, prostate hyperplasia, post-traumatic stress disorder, and scorpion stings (11). Its primaryknown mechanism is to antagonize α1-adrenergic receptors,a subfamily of GPCRs. GPCR receptor drugs often bind toGPCRs other than the primary target, and such off-targetinteractions can play important roles in therapy and toxicity.Here, we report an interesting off-target activity at DRD3.

ResultsPrazosin Inhibits Late Stages During Cell Division. In a screen forsmall-molecule inhibitors of cytokinesis (12), the final step of celldivision, Prazosin (Fig. 1A) unexpectedly scored as a strong hit,with over 80% of dividing HeLa cells becoming binucleated (Fig.1 B and C). Our initial screen was in Drosophila Kc167 cells, butwe found that the actions of Prazosin were similar in all mam-malian cell lines tested (Fig. 1D), suggesting a fairly generalmechanism. A chemically related compound, Terazosin (Fig.1A), was inactive and is used as a control throughout this work.Because Terazosin antagonizes α1-adrenergic receptors as ef-fectively as Prazosin, the effect of Prazosin on cytokinesis is likelyto be an off-target interaction. Time-lapse imaging showed thatPrazosin blocks cytokinesis at the abscission stage after furrowconstriction (SI Appendix, SI Materials and Methods and Fig. S1).Abscission is thought to require complex plasma membrane dy-namics, including secretion and endocytosis (13), which suggeststhat Prazosin might perturb these dynamics.

Prazosin Induces Endosomal Tubules and Inhibits Endosomal Sorting.EM analysis revealed that Prazosin treatment induced strikingmembrane tubules within the cytoplasm up to 20 μm in lengthand ∼100 nm in diameter (Fig. 2A and SI Appendix, SI Materialsand Methods). These tubules morphologically resembled onereported form of early endosomes (7). Prazosin-induced tubuleswere strongly labeled by fluorescent transferrin, a marker ofendocytic trafficking (Fig. 2B), and gold nanoparticles coupled totransferrin receptor antibodies localized to tubules (Fig. 2A).Robust transferrin-stained tubules formed within 10 min of20 μM Prazosin treatment and were present in nearly 100% ofcells, indicating a lack of cell cycle dependence. The effect wasreversible; tubules disappeared within minutes after drug wash-out (Fig. 2C and SI Appendix, Fig. S2A).

Author contributions: X.Z., W.W., M.L.C., T.J.M., and U.S.E. designed research; X.Z., W.W.,A.V.B., and M.L.C. performed research; X.Z., W.W., A.V.B., M.L.C., T.J.M., and U.S.E. ana-lyzed data; and X.Z., T.J.M., and U.S.E. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.1To whom correspondence should be addressed. E-mail: ulrike.eggert@kcl.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1207821109/-/DCSupplemental.

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Prazosin perturbed a specific step in the endocytic pathway.Initial vesicle internalization from the plasma membrane was notperturbed by Prazosin (SI Appendix, Fig. S3A). Localization ofclathrin (SI Appendix, Fig. S3B) was not perturbed, and lyso-some-associated membrane protein (LAMP1) (a marker oflysosomes) (SI Appendix, Fig. S3B) or fluorescently labeled EGFresident in lysosomes after pulse-chase experiments were notperturbed (Fig. 2B). Endoplasmic reticulum or Golgi residentproteins were also not affected (SI Appendix, Fig. S3B), andendosomal pH was not affected. Recycling endosome markers(transferrin, transferrin receptor, and Rab11) showed robuststaining along the tubules along with sorting nexin 1 (SNX1)(Fig. 2D and SI Appendix, Fig. S2B) and proteins that trafficbetween endosomes and the Golgi, such as cation-independentmannose 6 phosphate receptor (CI-M6PR) (Fig. 2E). Prazosin-induced tubules contain patches of early endosomal markers,a portion of which resided stably in tubules while the rest existedas vesicles (Fig. 2D and SI Appendix, Fig. S2B).To test if Prazosin inhibits endosomal sorting, we added two

differentially labeled cargoes, EGF and transferrin, to live cells.In untreated cells, both were trafficked through the endocyticpathway; however, EGF then moved to lysosomes, and trans-ferrin moved to recycling endosomes and eventually, the plasmamembrane as expected (14). We showed, in Fig. 2B, that EGFalready resident in lysosomes is not affected by Prazosin. How-ever, EGF localized to and remained in tubules if it was added tocells pretreated with Prazosin; transferrin did the same, and

recycling was inhibited by Prazosin (Fig. 2 F–H and SI Appendix,SI Materials and Methods). Unlike Brefeldin, which inducestransferrin-positive endosomal tubules without blocking sorting(Fig. 2 F and G), Prazosin seems to be an inhibitor ofendosomal sorting.Some endosomal pathways use microtubules as highways to

transport vesicles to cellular locations as required. Early steps ofsorting are thought to be independent of microtubules (15–18),but the morphology of the membrane tubules suggested a possi-ble cytoskeleton involvement. We found that Prazosin-inducedtubules are not much affected by microtubule depolymerization(SI Appendix, Fig. S4 A and B). Prazosin-induced tubules werealso independent of actin (SI Appendix, Fig. S4C). To ourknowledge, Prazosin treatment is the only type of cellular per-turbation that can cause complete but reversible tubulation ofsorting endosomes, providing an opportunity to study the factorsthat regulate the formation and turnover of these importanttrafficking platforms.

Effects of Prazosin on Endocytic Sorting Are Independent of ItsClinical Targets, the Adrenergic Receptors. To determine if theadrenergic receptors are involved in Prazosin-induced endosometubulation, we used RNAi to deplete α1-adrenergic receptors inHeLa cells, which did not inhibit their response to Prazosin (SIAppendix, Fig. S5). Other small-molecule adrenergic antagonists,either closely related to Prazosin (e.g., Terazosin) (Fig. 1A) orchemically unrelated (e.g., Corynanthine), did not cause thesame phenotypes as Prazosin, even at high concentrations (SIAppendix, Fig. S5).

Effects of Prazosin on Endocytic Sorting Are Mediated by DRD3.Drugs that target GPCRs can be promiscuous in binding activitybetween related GPCRs. We profiled the activity of Prazosinacross a GPCR panel using functional readouts and found that,in addition to adrenergic receptors, it robustly antagonized 5 of158 GPCRs tested (SI Appendix, Table S1A), including dopa-mine receptors D1 and D2. RNAi knockdown of these receptorsin HeLa did not significantly affect the Prazosin response. Un-expectedly, however, RNAi of the related DRD3, which was notin the GPCR panel, completely blocked the endosome tubula-tion activity of Prazosin (Fig. 3 A and B) as well as its cytokinesis-inhibiting activity (Fig. 3C). Unlike other dopamine receptorsthat are significantly enriched in the brain, DRD3 seems to beexpressed at low levels in most tissues (www.genevestigator.com) as well as HeLa cells (Fig. 3B and SI Appendix, SI Materialsand Methods) (19) and other cancer cells (20), suggesting that itmight play a broader role. To confirm that our observations werenot caused by off-target effects of the siRNAs targeting this re-ceptor, we constructed a cell line that expressed an RNAi-re-sistant GFP-DRD3 allele (SI Appendix, SI Materials andMethods). As expected for a specific knockdown, DRD3 RNAi inthe RNAi-resistant cell line did not inhibit the effects of Prazosinon tubulation and cytokinesis (Fig. 3D).Using a standard downstream signaling (cAMP level) assay

and a β-Arrestin Recruitment Tango Assay (21) (SI Appendix, SIMaterials and Methods), we tested if Prazosin acts on DRD3 ina classical manner. We found that it did not behave as a classicalGPCR agonist, antagonist, or allosteric modulator (SI Appendix,Fig. S6 and Table S1B). This finding is consistent with our ob-servation that treatment with other DRD3 agonists/antagonists,including 7-OH-DPAT and SB-27701-A, does not give rise to thesame tubulation, inhibition of transferrin recycling, or cytokinesisblock phenotypes as Prazosin (SI Appendix, Fig. S7). This findingsuggests that Prazosin does not act at traditional small-moleculebinding sites on DRD3.

Knockdown of DRD3 Affects Endocytic Sorting Independently ofPrazosin. To test if DRD3 regulates endocytic sorting in-dependent of Prazosin action, we evaluated the effect of DRD3RNAi and found that DRD3 knockdown alone shares some ofthe effects of Prazosin on cells. Like Prazosin, DRD3 knockdown

Fig. 1. Prazosin inhibits cytokinesis. (A) Chemical structures of Prazosin andits inactive analog Terazosin. (B) Fixed cell analysis shows that Prazosininduces cytokinesis failure and binucleated cell formation, whereas Ter-azosin does not. HeLa cells were incubated with DMSO, 20 μM Prazosin, orTerazosin for 36 h before being fixed and stained for microtubules (red)and DNA (white). Binucleated cells are indicated by an asterisk. (Scale bar:10 μm.) (C) Quantification of B. (D) Quantification of cytokinesis failure indifferent cell lines after Prazosin treatment shows that Prazosin-inducedcytokinesis inhibition is conserved. Terazosin-treated HeLa cells are also in-cluded (SI Appendix, SI Materials and Methods).

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did not affect initial internalization of fluorescent transferrin orEGF (SI Appendix, Figs. S3A and S8A) or localization of clathrinor dynamin (SI Appendix, Figs. S3B and S8B). However, bothperturbed transferrin trafficking, and DRD3 knockdown causeda modest increase in binucleated cells (two- to threefold) (Fig.3C and SI Appendix, Fig. S8C). Trafficking between endosomesand the Golgi, as visualized by CI-M6PR staining, was alsoperturbed by both treatments (Figs. 2E and 4A). DRD3 knock-down caused an overall reduction of transferrin staining, and theremaining vesicles were enlarged (Figs. 3A and 4B), which can bea consequence of endocytic sorting defects. To test if endocyticsorting was affected by the absence of DRD3, we treated cellswith a pulse of fluorescently labeled markers (transferrin, EGF,and dextran) and observed their progress through the endocytictransport network after chasing with unlabeled medium as de-scribed above for Prazosin. In control and DRD3 RNAi cells,most vesicles contained all three markers 20 min after treatment(Fig. 4 B and C), suggesting that they had reached sortingendosomes and were being prepared for transport to their re-spective pathways; 80 min after chasing in control cells, themarkers had separated almost completely, and each marker hadarrived at its appropriate destination (Fig. 4 B and C). In con-trast and similar to Prazosin treatment, the fluorescent markerscontinued to be highly colocalized at 80 min in DRD3-depleted

cells (Fig. 4 B and C), and many of these endosomes were pos-itive for a sorting endosome marker, SNX1 (Fig. 4D). Alsosimilar to Prazosin treatment, transferrin recycling, measured byFACS analysis, was inhibited in DRD3 knockdown cells (Fig.4E). Thus, loss of dopamine receptor DRD3 seems to slow downendocytic sorting.

Prazosin Stabilizes an Interaction Between DRD3 and the CoatomerComplex COPI.Our data suggest that either Prazosin binds directlyto DRD3 in a nonclassical way or DRD3 is required to manifestthe effects of Prazosin binding to another target. We assayed theeffects of Prazosin on interactions of other proteins with DRD3using immunoprecipitation from cell lines stably expressingFLAG-tagged GFP-DRD3 (22) (SI Appendix, SI Materials andMethods). We focused on interaction partners likely to mediatemembrane dynamics. Although the most obvious candidates,including dynamin and sorting nexins 1 and 2, showed not effect,Prazosin substantially increased the interaction between DRD3and subunits of the COPI coatomer complex. We tested COPI,because it is involved in membrane trafficking and was previouslyassociated with cytokinesis failure (12). All complex members forwhich we could obtain antibodies were strongly enriched in theDRD3 pull-down assay but only in the presence of Prazosin(Fig. 5A).

Fig. 2. Prazosin induces endosomal tubulation and inhibits sorting. (A) Electron micrographs of Prazosin-induced transferrin receptor positive tubules.Transferrin receptor antibody-coupled gold beads were added to HeLa cells for 10 min before control DMSO, or 30 μM Prazosin was added for 1 additional h.Red arrows show gold beads in vesicles or tubules. (Scale bar: 200 nm.) (B) Prazosin-treated HeLa cells exhibit robust transferrin receptor-positive endosomaltubules, whereas lysosomes are not affected. Alexa488-Transferrin and Alexa555-EGF were added to cells for 45 min. Then, cells were chased in the presenceof DMSO or 30 μM Prazosin for 30 min in marker-free medium to allow EGF to reach the lysosomes and clear from earlier pathways. (C) Quantification of theendosomal tubules in Prazosin-treated cells and washout cells using fixed cell analysis. Cells were treated with DMSO or 30 μM Prazosin for 30 min followed bywashing out with drug-free medium for 30 min. Endosomal tubules longer than 5 μm are classified as long endosomal tubules. (D) Prazosin induces an arrayof endosomal tubules that contain transferrin (and transferrin receptor and Rab11) (SI Appendix, Fig. S2B), SNX1 (and SNX2), and EEA1 (and Rab5) (SI Ap-pendix, Fig. S2B). Representative micrographs show transferrin, SNX1, and EEA1 localization in a Prazosin-treated HeLa cell. Alexa 488-Transferrin (green) wasadded to HeLa cells for 10 min before 30 μM Prazosin was added for an additional 1 h. Cells were washed and fixed before staining with anti-SNX1 (red) andanti-EEA1 (blue) antibodies. (E) Prazosin-treated HeLa cells form tubules that contain CI-M6PR. (F) Endosomal sorting is disrupted in endosomal tubulesinduced by Prazosin but not Brefeldin A. Pulse and chase experiments in HeLa cells show that both transferrin and EGF are trapped in endosomal tubulesduring sorting. HeLa cells were treated with DMSO control, 30 μM Prazosin, or 10 μg/mL BFA for 1 h. Then, Alexa 488-Transferrin and Alexa 555-EGF wereadded for 10 min. Images were taken at indicated times after washing out with marker- and phenol red-free medium. (G) Quantification of results in F.(H) Flow cytometry analysis of transferrin recycling in control, 30 μM Prazosin, or Terazosin-treated HeLa cells.

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The COPI complex is important in retrograde transport fromthe Golgi to the endoplasmic reticulum (23, 24), and a role in thebudding of vesicles from early endosomes has also been pro-posed (25–30). In untreated cells, COPI subunits localizedmostly to the Golgi, and a small fraction localized to the cyto-plasm or vesicles distributed throughout the cytoplasm. Prazosintreatment caused an increased fraction of COPI subunits todissociate from the Golgi and localize to cytoplasmic non-Golgiregions (Fig. 5B). We previously reported that COPI RNAiresults in cytokinesis failure, but generally, RNAi of most COPIsubunits is cytotoxic, making it difficult to assess their functions(12). Although knockdown of either isoform of β-COP (COPB1and COPB2) is also toxic to an extent, the remaining live cellsshow similar phenotypes to DRD3 RNAi and prevent the for-mation of Prazosin-induced endosomal tubules (Fig. 5C). Thisfinding indicates that COPI may be required for the effect ofPrazosin on endosomes and cytokinesis. β-COP knockdown inthe absence of Prazosin also inhibited endosomal sorting andtransferrin recycling in a manner similar to DRD3 knockdown(Fig. 5 D and E).GPCRs can bind effector proteins through their cytoplasmic

loops, and the third loop has been implicated in dopaminereceptors’ interactions with multiple binding partners (31, 32).

We constructed a cell line expressing DRD3-RNAi–resistantGFP-DRD3ΔL-FLAG, where the third cytoplasmic loop wasreplaced with the shorter second cytoplasmic loop (SI Appendix,SI Materials and Methods). Prazosin-induced tubule formationwas prevented in cells where the loop mutant replaced endoge-nous DRD3 (SI Appendix, Fig. S9A). Furthermore, COPI bind-ing to DRD3 in the presence of Prazosin was reduced in cellsexpressing GFP-DRD3ΔL-FLAG (SI Appendix, Fig. S9B). Al-though these data suggest that the third cytoplasmic loop ofDRD3 is involved in the effects of Prazosin, we cannot excludethe possibility that the mutant is incorrectly folded and therefore,unable to rescue the WT response to Prazosin. Understandinghow interactions between DRD3 and COPI contribute toendosome conformational changes and sorting defects is a highpriority in the future.

DiscussionOur experiments implicate DRD3 and COPI in endocytic sort-ing. Individual removal of either DRD3 or COPI proteins leadsto sorting defects (Figs. 4 B–D and 5D). However, neither ofthese treatments by themselves result in the formation oftubules, and both treatments prevent tubule formation in re-sponse to Prazosin. These data support a model in which DRD3

Fig. 3. DRD3 is involved in endocytic sorting and mediates the effects of Prazosin. (A) DRD3 RNAi prevents Prazosin-induced endosomal tubule formation.Transferrin receptor staining is shown. HeLa cells were treated with control or DRD3 siRNA for 3 d before DMSO or 30 μM Prazosin was added for 1 h. (B)Western blot and semiquantitative RT-PCR show the expression and knockdown of DRD3 in HeLa cells. (C) DRD3 RNAi causes an increase in binucleated cellsand prevents strong cytokinesis inhibition induced by Prazosin. (D) Western blots show the specificity and efficiency of DRD3 RNAi knockdown. Cells with longendosomal tubules (longer than 5 μm) were counted in three independent experiments. (E) The localization of DRD3. GFP-DRD3-FLAG-HeLa cells were treatedwith control DMSO or 30 μM Prazosin for 1 h before being fixed in PBS + formaldehyde. Cells were processed with anti-GFP antibody staining using twodifferent conditions. Left shows cells that were permeabilized using 0.1% Triton X-100, and Right shows unpermeablized cells. Live GFP-DRD3-FLAG-HeLa cellsshow similar localizations to the anti-GFP staining in permeablized cells, indicating that the membrane population of DRD3 is relatively low compared with itsintracellular population, which is similar to transferrin receptor. The commercially available anti-DRD3 antibody shows highly unspecific staining and is notsuitable for immunofluorescence. (Scale bar: 10 μm.)

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and COPI both function in sorting and the addition of Prazosinstabilizes a nonproductive complex between them and probably,other proteins (Fig. 5A). The concept of a small-molecule sta-bilizing interactions between proteins is familiar from chemicalinducers of dimerization (33), suggesting that Prazosin mayact similarly.Although our data suggest that the third cytoplasmic loop of

D3 might be involved, exactly where Prazosin binds in thiscomplex is not yet clear. The fact that it antagonizes DRD1 and-2 suggests that it can bind to closely related GPCRs and may,therefore, bind to DRD3 directly. Perhaps it binds to DRD3 ina nonstandard manner that is not competitive with traditional

ligands, or perhaps it induces a conformational change, possiblyperturbing receptor oligomerization, which is increasingly rec-ognized as a key step in GPCR function (34). The very sharpdose–response curve observed for Prazosin (Fig. 1D) couldsupport an oligomerization model, where a signaling cascade istriggered when a critical mass has been reached.Although their ultimate effects on cells are different (e.g.,

Brefeldin perturbs Golgi, but Prazosin does not; morphology andstability of Prazosin- and Brefeldin-induced endosome tubulesare different) (Fig. 2F and SI Appendix, Fig. S4), the mechanismsof action of Prazosin and Brefeldin involve the same proteincomplex, the coatomer COPI. Brefeldin has been used success-fully to study Golgi dynamics (10). COPI localizes not only to the

Fig. 4. DRD3 is involved in endocytic sorting. (A) DRD3 RNAi causes in-creased CI-M6PR localization to vesicles in addition to the Golgi. Control orDRD3 RNAi-treated cells were stained for CI-M6PR (green) and a residentGolgi marker GM130 (red). (B) Pulse and chase experiments show sortingdefects in DRD3 RNAi cells. HeLa cells were treated with control or DRD3RNAi for 3 d before Alexa 555-EGF, FITC-70 kD-dextran, and Alexa 647-Transferrin were added for 10 min followed by washing out with marker-and phenol red-free medium. Pictures were taken at indicated time points inlive cells. (C) Quantification of EGF and/or dextran that colocalize or partiallycolocalize with transferrin; (D) Endocytic cargoes are trapped in SNX1-posi-tive endosomes after DRD3 RNAi. HeLa cells were treated with control orDRD3 RNAi for 3 d before Alexa 555-EGF (red) was added for 10 min fol-lowed by washing out with marker-free medium for indicated time points.Cells were than washed, fixed, and stained with anti-SNX1 (green). EGF thatcolocalizes or partially colocalizes with SNX1 was quantified. For each con-dition, around 50 cells were counted. Mean values from two independentexperiments are shown. (Scale bar: 10 μm.) (E) Flow cytometry analysisof transferrin recycling in control or DRD3 RNAi knockdown HeLa cells.

Fig. 5. The COPI complex interacts with DRD3 in the presence of Prazosinand is involved in its effects on endocytosis. (A) Pull-down experiments usinganti-FLAG antibody in GFP-DRD3-FLAG-HeLa cells treated with DMSO, Pra-zosin, or Terazosin show that the interaction of DRD3 with COPI subunitsCOPB, COPC, and COPG is increased after Prazosin treatment. (B) COPB,COPD, and COPG localizations are disrupted by Prazosin treatment. HeLacells were treated with DMSO or 30 μM Prazosin for 1 h before fixing andstaining with COP antibodies. (C) COPB1 or COPB2 RNAi prevents endosomaltubule formation in Prazosin-treated HeLa cells. Transferrin receptor stain-ing is shown. HeLa cells were treated with siRNAs for 3 d before DMSO or 30μM Prazosin was added for 1 h. (D) Pulse and chase experiments showsorting defects in COPB RNAi cells. HeLa cells were treated with control orCOPB1 + COPB2 RNAi for 3 d before Alexa 488-Tf and FITC-70 kD-dextranwere added for 10 min followed by washing out with marker- and phenolred-free medium. Individual COPB1 or COPB2 RNAi resulted in similar phe-notypes. Pictures were taken at indicated time points without fixing thecells. (Scale bar: 10 μm.) (E) Flow cytometry analysis of transferrin recycling incontrol or COPB1 or COPB2 RNAi knockdown HeLa cells.

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Golgi but also to small vesicles and the cytoplasm. Similarly,DRD3 is also found in different cellular pools (Fig. 3E). Prazosinseems to target specific subpools of both proteins. It triggers anincreased association between DRD3 and COPI and their absencefrom the newly formed endosomal tubules. Taken together, thesedata suggest that a transient but important regulatory interactionbetween DRD3 and COPI, stabilized by Prazosin, is key in regu-lating the equilibrium between vesicular and tubular endosomesand therefore, endosomal sorting.The effect of Prazosin on endosome dynamics is interesting

clinically, both because endosomal trafficking is involved in nu-merous disease-related processes and because the action ofPrazosin implicates a GPCR from an important family in a basicand general biological regulatory process. Given that manyclinically approved drugs targets GPCRs, a better understandingof these receptors’ biological functions will be critical in de-veloping more efficacious therapeutics. Prazosin is the onlyclinically approved drug that inhibits a specific step during en-docytosis, and we know from its long history that it is relativelynontoxic. Targeting endocytic processes clinically would requirehigher Prazosin doses than those doses currently used in anti-hypertension therapy, because concentrations in patients’ bloodare about 100-fold less than we reported here; however, Prazosincan be a starting point in additional development. Our findingshighlight the increasingly accepted concept that even the mostwidely used and safe drugs often have unexpected targets andmechanisms. Off-target effects of compounds that have beenthoroughly vetted in humans are always of great interest to bothunderstand their efficacy and toxicity in their current indicationsand spark ideas for new indications.

Materials and MethodsCell Culture and Immunofluorescence. HeLa cells were grown in monolayers inDMEM supplemented with 10% (vol/vol) heat-inactivated FBS (Invitrogen)

and 1% (vol/vol) penicillin/streptomycin (P/S; Cellgro). Prazosin (P7791) andTerazosin (T4680) are from Sigma.

For all immunofluorescence experiments, cells were fixed in either 3.7%(vol/vol) formaldehyde in PBS for 20 min or −20 °C methanol for 5 min,permeabilized with 0.1% Triton X-100 in TBS, blocked in AbDil (0.1% TritonX-100 in TBS + 2% (wt/vol) BSA + 0.1% NaN3), and probed with primary andsecondary antibodies diluted in AbDil. Cells were stained with DAPI andmounted in Prolong gold mounting medium (Invitrogen).

The following antibodies and markers were used in immunofluorescenceand Western blots: anti-GFP, Transferrin receptor, LAMP1, Clathrin, CI-M6PR,GM130, Giantin, COPB and COPD antibodies (Abcam), EEA1, SNX1 and SNX2antibodies (BD Bioscience), TRAPα antibody (a gift from Tom Rapoport,Harvard Medical School, Boston, MA), DRD3 antibody (Calbiochem), fluo-rescently labeled transferrin and EGF (Invitrogen), FITC-70 kD dextran andmouse anti-FLAG antibody (Sigma), and COPG antibody (Santa Cruz).

Statistics. For quantifications in themanuscript, mean values are shown in thefigures, and SDs are shown as error bars. All images shown in figures arerepresentative. 500 (Fig. 1C), 300 (Figs. 2C, 3 C and D, and 5C), or 50 (Figs. 2Gand 4C) cells were counted for each condition in three independentexperiments. Comparisons between treatments were analyzed by a two-tailed Student t test. P values are labeled in the figures for where data werecompared. In Figs 2H, 4E, and 5E, 10,000 cells each were collected in threeindependent experiments. Mean fluorescence intensities normalized to0 min are shown.

ACKNOWLEDGMENTS. We thank the Nikon Imaging Center at HarvardMedical School for assistance with microscopy, the Flow Cytometry facility atthe Dana-Farber Cancer Institute for assistance with flow cytometry, andQingsong Liu and members of the U.S.E. laboratory for helpful discussions.We also thank Maria F. Sassano and Bryan L. Roth at the University of NorthCarolina and the National Institute of Mental Health Psychoactive DrugScreening Program for conducting the experiments shown in SI Appendix,Fig. S6. M.L.C. and T.J.M. were supported by National Institutes of HealthGrant R01 GM023928. This project was funded by National Institutes ofHealth Grant R01 GM082834 (to U.S.E.) and the Dana-Farber Cancer Institute.

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