Dopamine Receptor D3 Regulates Endocytic Sorting by a Prazosin-Sensitive Interaction with the Coatomer COPI

Dopamine receptor D3 regulates endocytic sorting by a Prazosin-sensitive interaction with the coatomer COPI

Xin 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 Medical School, Boston, MA 02115; cHigh Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, 230031, People’s Republic of China; and eDepartment of Chemistry 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 by endocytosis and are sorted to different Prazosin, which we describe here as a tool for endocytosis cellular destinations in early/sorting endosomes. The mechanism and research, is an important drug that has been used clinically for regulation of sorting are poorly understood, although transitions decades to treat hypertension, prostate hyperplasia, post- between vesicular and tubular endosomes are important. We found traumatic stress disorder, and scorpion stings (11). Its primary that the antihypertensive drug Prazosin inhibits endocytic sorting by known mechanism is to antagonize α1-adrenergic receptors, an off-target perturbation of the G protein-coupled receptor dopa- a subfamily of GPCRs. GPCR receptor drugs often bind to

mine receptor D3 (DRD3). Prazosin is also a potent cytokinesis inhib- GPCRs other than the primary target, and such off-target itor, likely as a consequence of its effects on endosomes. Prazosin interactions can play important roles in therapy and toxicity. stabilizes a normally transient interaction between DRD3 and the Here, we report an interesting off-target activity at DRD3. coatomer COPI, a complex involved in membrane transport, and shifts endosomal morphology entirely to tubules, disrupting cargo Results sorting. RNAi depletion of DRD3 alone also inhibits endocytic sorting, Prazosin Inhibits Late Stages During Cell Division. In a screen for indicating a noncanonical role for a G protein-coupled receptor. small-molecule inhibitors of cytokinesis (12), the ﬁnal step of cell Prazosin is a powerful tool for rapid and reversible perturbation division, Prazosin (Fig. 1A) unexpectedly scored as a strong hit, of endocytic dynamics. with over 80% of dividing HeLa cells becoming binucleated (Fig. 1 B and C). Our initial screen was in Drosophila Kc167 cells, but small-molecule inhibitor | endocytic tubulation | we found that the actions of Prazosin were similar in all mam- unconventional G protein-coupled receptor function | malian cell lines tested (Fig. 1D), suggesting a fairly general drug off-target effects | membrane trafﬁcking mechanism. A chemically related compound, Terazosin (Fig. 1A), was inactive and is used as a control throughout this work. α protein-coupled receptors (GPCRs) are the most important Because Terazosin antagonizes 1-adrenergic receptors as ef- Gchemosensing receptors in animals and the targets of many fectively as Prazosin, the effect of Prazosin on cytokinesis is likely therapeutic drugs. They have mostly been studied from the to be an off-target interaction. Time-lapse imaging showed that perspective of signal transduction and pharmacology, but there Prazosin blocks cytokinesis at the abscission stage after furrow have been hints that GPCRs are important regulators of basic constriction (SI Appendix, SI Materials and Methods and Fig. S1). cellular processes (1, 2). The dopamine receptors (D1–5) are Abscission is thought to require complex plasma membrane dy- GPCRs with important functions in the brain, and they are tar- namics, including secretion and endocytosis (13), which suggests

geted by numerous drugs used to treat neurological disorders that Prazosin might perturb these dynamics. BIOCHEMISTRY ranging from Parkinson disease to schizophrenia. Although there is a large amount of literature about the regulation of GPCR Prazosin Induces Endosomal Tubules and Inhibits Endosomal Sorting. signaling by endocytosis, much less is known about how or if EM analysis revealed that Prazosin treatment induced striking GPCRs, in turn, regulate endocytosis (3–5). Our data show that membrane tubules within the cytoplasm up to 20 μm in length the dopamine receptor D3 (DRD3) plays an unexpected role in and ∼100 nm in diameter (Fig. 2A and SI Appendix, SI Materials endocytic sorting. and Methods). These tubules morphologically resembled one Many different proteins and complexes enter cells by endo- reported form of early endosomes (7). Prazosin-induced tubules cytosis, and they must be rapidly sorted for transport to different were strongly labeled by fluorescent transferrin, a marker of locations in the cell. For example, some cargoes are recycled to endocytic trafficking (Fig. 2B), and gold nanoparticles coupled to the plasma membrane, whereas others are sent to lysosomes. transferrin receptor antibodies localized to tubules (Fig. 2A). Sorting occurs in specialized compartments called early or sort- Robust transferrin-stained tubules formed within 10 min of ing endosomes. Highly dynamic trafficking occurs between these 20 μM Prazosin treatment and were present in nearly 100% of compartments and multiple other cellular compartments, driven by sorting, budding, fission, and fusion reactions (6). Although cells, indicating a lack of cell cycle dependence. The effect was reversible; tubules disappeared within minutes after drug wash- some individual steps in endocytic sorting have been elucidated, C SI Appendix A their coordination in cells remains mysterious. Dynamic tran- out (Fig. 2 and , Fig. S2 ). sitions between vesicular and tubular endosomes seem to be key factors in determining the fate of endocytic cargoes; the coatomer complex COPI may play a role in these dynamics (7, 8), but Author contributions: X.Z., W.W., M.L.C., T.J.M., and U.S.E. designed research; X.Z., W.W., precisely how these transitions are regulated is unclear. One 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- reason that it has been difficult to elucidate sorting endosome lyzed data; and X.Z., T.J.M., and U.S.E. wrote the paper. dynamics in living cells has been the lack of small-molecule tools The authors declare no conflict of interest. to rapidly and reversibly perturb them. Elucidation of Golgi *This Direct Submission article had a prearranged editor. dynamics benefitted greatly from use of Brefeldin (9), a small 1To whom correspondence should be addressed. E-mail: [email protected]. molecule that inhibits Arf guanine-nucleotide exchange factor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (ArfGEF), perturbing the functions of COPI at the Golgi (10). 1073/pnas.1207821109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1207821109 PNAS | July 31, 2012 | vol. 109 | no. 31 | 12485–12490 Downloaded by guest on September 29, 2021 recycling was inhibited by Prazosin (Fig. 2 F–H and SI Appendix, SI Materials and Methods). Unlike Brefeldin, which induces transferrin-positive endosomal tubules without blocking sorting (Fig. 2 F and G), Prazosin seems to be an inhibitor of endosomal sorting. Some endosomal pathways use microtubules as highways to transport vesicles to cellular locations as required. Early steps of sorting 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-induced tubules are not much affected by microtubule depolymerization (SI Appendix, Fig. S4 A and B). Prazosin-induced tubules were also independent of actin (SI Appendix, Fig. S4C). To our knowledge, Prazosin treatment is the only type of cellular perturbation that can cause complete but reversible tubulation of sorting endosomes, providing an opportunity to study the factors that regulate the formation and turnover of these important trafﬁcking platforms.

Effects of Prazosin on Endocytic Sorting Are Independent of Its Clinical Targets, the Adrenergic Receptors. To determine if the adrenergic receptors are involved in Prazosin-induced endosome tubulation, we used RNAi to deplete α1-adrenergic receptors in HeLa cells, which did not inhibit their response to Prazosin (SI Appendix, Fig. S5). Other small-molecule adrenergic antagonists, either closely related to Prazosin (e.g., Terazosin) (Fig. 1A)or chemically unrelated (e.g., Corynanthine), did not cause the same phenotypes as Prazosin, even at high concentrations (SI Appendix, Fig. S5).

Effects of Prazosin on Endocytic Sorting Are Mediated by DRD3. Drugs that target GPCRs can be promiscuous in binding activity fi Fig. 1. Prazosin inhibits cytokinesis. (A) Chemical structures of Prazosin and between related GPCRs. We pro led the activity of Prazosin its inactive analog Terazosin. (B) Fixed cell analysis shows that Prazosin across a GPCR panel using functional readouts and found that, in addition to adrenergic receptors, it robustly antagonized 5 of induces cytokinesis failure and binucleated cell formation, whereas Ter- SI Appendix A azosin does not. HeLa cells were incubated with DMSO, 20 μM Prazosin, or 158 GPCRs tested ( , Table S1 ), including dopa- Terazosin for 36 h before being fixed and stained for microtubules (red) mine receptors D1 and D2. RNAi knockdown of these receptors and DNA (white). Binucleated cells are indicated by an asterisk. (Scale bar: in HeLa did not significantly affect the Prazosin response. Un- 10 μm.) (C) Quantification of B.(D) Quantification of cytokinesis failure in expectedly, however, RNAi of the related DRD3, which was not different cell lines after Prazosin treatment shows that Prazosin-induced in the GPCR panel, completely blocked the endosome tubula- cytokinesis inhibition is conserved. Terazosin-treated HeLa cells are also in- tion activity of Prazosin (Fig. 3 A and B) as well as its cytokinesis- cluded (SI Appendix, SI Materials and Methods). inhibiting activity (Fig. 3C). Unlike other dopamine receptors that are significantly enriched in the brain, DRD3 seems to be expressed at low levels in most tissues (www.genevestigator. Prazosin perturbed a specific step in the endocytic pathway. com) as well as HeLa cells (Fig. 3B and SI Appendix, SI Materials Initial vesicle internalization from the plasma membrane was not and Methods) (19) and other cancer cells (20), suggesting that it perturbed by Prazosin (SI Appendix, Fig. S3A). Localization of might play a broader role. To confirm that our observations were clathrin (SI Appendix, Fig. S3B) was not perturbed, and lyso- not caused by off-target effects of the siRNAs targeting this re- some-associated membrane protein (LAMP1) (a marker of ceptor, we constructed a cell line that expressed an RNAi-re- lysosomes) (SI Appendix, Fig. S3B)orfluorescently labeled EGF sistant GFP-DRD3 allele (SI Appendix, SI Materials and resident in lysosomes after pulse-chase experiments were not Methods). As expected for a specific knockdown, DRD3 RNAi in perturbed (Fig. 2B). Endoplasmic reticulum or Golgi resident the RNAi-resistant cell line did not inhibit the effects of Prazosin proteins were also not affected (SI Appendix, Fig. S3B), and on tubulation and cytokinesis (Fig. 3D). endosomal pH was not affected. Recycling endosome markers Using a standard downstream signaling (cAMP level) assay (transferrin, transferrin receptor, and Rab11) showed robust and a β-Arrestin Recruitment Tango Assay (21) (SI Appendix, SI staining along the tubules along with sorting nexin 1 (SNX1) Materials and Methods), we tested if Prazosin acts on DRD3 in (Fig. 2D and SI Appendix, Fig. S2B) and proteins that traffic a classical manner. We found that it did not behave as a classical between endosomes and the Golgi, such as cation-independent GPCR agonist, antagonist, or allosteric modulator (SI Appendix, mannose 6 phosphate receptor (CI-M6PR) (Fig. 2E). Prazosin- Fig. S6 and Table S1B). This finding is consistent with our ob- induced tubules contain patches of early endosomal markers, servation that treatment with other DRD3 agonists/antagonists, a portion of which resided stably in tubules while the rest existed including 7-OH-DPAT and SB-27701-A, does not give rise to the as vesicles (Fig. 2D and SI Appendix, Fig. S2B). same tubulation, inhibition of transferrin recycling, or cytokinesis To test if Prazosin inhibits endosomal sorting, we added two block phenotypes as Prazosin (SI Appendix, Fig. S7). This finding differentially labeled cargoes, EGF and transferrin, to live cells. suggests that Prazosin does not act at traditional small-molecule In untreated cells, both were trafficked through the endocytic binding sites on DRD3. pathway; however, EGF then moved to lysosomes, and transferrin moved to recycling endosomes and eventually, the plasma Knockdown of DRD3 Affects Endocytic Sorting Independently of membrane as expected (14). We showed, in Fig. 2B, that EGF Prazosin. To test if DRD3 regulates endocytic sorting in- already resident in lysosomes is not affected by Prazosin. How- dependent of Prazosin action, we evaluated the effect of DRD3 ever, EGF localized to and remained in tubules if it was added to RNAi and found that DRD3 knockdown alone shares some of cells pretreated with Prazosin; transferrin did the same, and the effects of Prazosin on cells. Like Prazosin, DRD3 knockdown

12486 | www.pnas.org/cgi/doi/10.1073/pnas.1207821109 Zhang et al. Downloaded by guest on September 29, 2021 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 endosomal tubules, whereas lysosomes are not affected. Alexa488-Transferrin and Alexa555-EGF were added to cells for 45 min. Then, cells were chased in the presence of 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 the endosomal 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 by washing out with drug-free medium for 30 min. Endosomal tubules longer than 5 μm are classified as long endosomal tubules. (D) Prazosin induces an array of 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) was added 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) and anti-EEA1 (blue) antibodies. (E) Prazosin-treated HeLa cells form tubules that contain CI-M6PR. (F) Endosomal sorting is disrupted in endosomal tubules induced by Prazosin but not Brefeldin A. Pulse and chase experiments in HeLa cells show that both transferrin and EGF are trapped in endosomal tubules during 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 were added 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. BIOCHEMISTRY

did not affect initial internalization of fluorescent transferrin or cells (Fig. 4 B and C), and many of these endosomes were pos- EGF (SI Appendix, Figs. S3A and S8A) or localization of clathrin itive for a sorting endosome marker, SNX1 (Fig. 4D). Also or dynamin (SI Appendix, Figs. S3B and S8B). However, both similar to Prazosin treatment, transferrin recycling, measured by perturbed transferrin trafficking, and DRD3 knockdown caused FACS analysis, was inhibited in DRD3 knockdown cells (Fig. a modest increase in binucleated cells (two- to threefold) (Fig. 4E). Thus, loss of dopamine receptor DRD3 seems to slow down 3C and SI Appendix, Fig. S8C). Trafficking between endosomes endocytic sorting. and the Golgi, as visualized by CI-M6PR staining, was also perturbed by both treatments (Figs. 2E and 4A). DRD3 knock- Prazosin Stabilizes an Interaction Between DRD3 and the Coatomer down caused an overall reduction of transferrin staining, and the Complex COPI. Our data suggest that either Prazosin binds directly remaining vesicles were enlarged (Figs. 3A and 4B), which can be to DRD3 in a nonclassical way or DRD3 is required to manifest a consequence of endocytic sorting defects. To test if endocytic the effects of Prazosin binding to another target. We assayed the sorting was affected by the absence of DRD3, we treated cells effects of Prazosin on interactions of other proteins with DRD3 with a pulse of fluorescently labeled markers (transferrin, EGF, using immunoprecipitation from cell lines stably expressing and dextran) and observed their progress through the endocytic FLAG-tagged GFP-DRD3 (22) (SI Appendix, SI Materials and transport network after chasing with unlabeled medium as de- Methods). We focused on interaction partners likely to mediate scribed above for Prazosin. In control and DRD3 RNAi cells, membrane dynamics. Although the most obvious candidates, most vesicles contained all three markers 20 min after treatment including dynamin and sorting nexins 1 and 2, showed not effect, (Fig. 4 B and C), suggesting that they had reached sorting Prazosin substantially increased the interaction between DRD3 endosomes and were being prepared for transport to their re- and subunits of the COPI coatomer complex. We tested COPI, spective pathways; 80 min after chasing in control cells, the because it is involved in membrane trafficking and was previously markers had separated almost completely, and each marker had associated with cytokinesis failure (12). All complex members for arrived at its appropriate destination (Fig. 4 B and C). In con- which we could obtain antibodies were strongly enriched in the trast and similar to Prazosin treatment, the fluorescent markers DRD3 pull-down assay but only in the presence of Prazosin continued to be highly colocalized at 80 min in DRD3-depleted (Fig. 5A).

Zhang et al. PNAS | July 31, 2012 | vol. 109 | no. 31 | 12487 Downloaded by guest on September 29, 2021 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 cells and prevents strong cytokinesis inhibition induced by Prazosin. (D) Western blots show the specificity and efficiency of DRD3 RNAi knockdown. Cells with long endosomal tubules (longer than 5 μm) were counted in three independent experiments. (E) The localization of DRD3. GFP-DRD3-FLAG-HeLa cells were treated with control DMSO or 30 μM Prazosin for 1 h before being fixed in PBS + formaldehyde. Cells were processed with anti-GFP antibody staining using two different conditions. Left shows cells that were permeabilized using 0.1% Triton X-100, and Right shows unpermeablized cells. Live GFP-DRD3-FLAG-HeLa cells show similar localizations to the anti-GFP staining in permeablized cells, indicating that the membrane population of DRD3 is relatively low compared with its intracellular population, which is similar to transferrin receptor. The commercially available anti-DRD3 antibody shows highly unspecific staining and is not suitable for immunofluorescence. (Scale bar: 10 μm.)

The COPI complex is important in retrograde transport from We constructed a cell line expressing DRD3-RNAi–resistant the Golgi to the endoplasmic reticulum (23, 24), and a role in the GFP-DRD3ΔL-FLAG, where the third cytoplasmic loop was budding of vesicles from early endosomes has also been pro- replaced with the shorter second cytoplasmic loop (SI Appendix, posed (25–30). In untreated cells, COPI subunits localized SI Materials and Methods). Prazosin-induced tubule formation mostly to the Golgi, and a small fraction localized to the cyto- was prevented in cells where the loop mutant replaced endoge- plasm or vesicles distributed throughout the cytoplasm. Prazosin nous DRD3 (SI Appendix, Fig. S9A). Furthermore, COPI bind- treatment caused an increased fraction of COPI subunits to ing to DRD3 in the presence of Prazosin was reduced in cells dissociate from the Golgi and localize to cytoplasmic non-Golgi expressing GFP-DRD3ΔL-FLAG (SI Appendix, Fig. S9B). Al- regions (Fig. 5B). We previously reported that COPI RNAi though these data suggest that the third cytoplasmic loop of results in cytokinesis failure, but generally, RNAi of most COPI DRD3 is involved in the effects of Prazosin, we cannot exclude subunits is cytotoxic, making it difﬁcult to assess their functions the possibility that the mutant is incorrectly folded and therefore, (12). Although knockdown of either isoform of β-COP (COPB1 unable to rescue the WT response to Prazosin. Understanding and COPB2) is also toxic to an extent, the remaining live cells how interactions between DRD3 and COPI contribute to show similar phenotypes to DRD3 RNAi and prevent the for- endosome conformational changes and sorting defects is a high mation of Prazosin-induced endosomal tubules (Fig. 5C). This priority in the future. ﬁnding indicates that COPI may be required for the effect of Prazosin on endosomes and cytokinesis. β-COP knockdown in Discussion the absence of Prazosin also inhibited endosomal sorting and Our experiments implicate DRD3 and COPI in endocytic sort- transferrin recycling in a manner similar to DRD3 knockdown ing. Individual removal of either DRD3 or COPI proteins leads (Fig. 5 D and E). to sorting defects (Figs. 4 B–D and 5D). However, neither of GPCRs can bind effector proteins through their cytoplasmic these treatments by themselves result in the formation of loops, and the third loop has been implicated in dopamine tubules, and both treatments prevent tubule formation in re- receptors’ interactions with multiple binding partners (31, 32). sponse to Prazosin. These data support a model in which DRD3

12488 | www.pnas.org/cgi/doi/10.1073/pnas.1207821109 Zhang et al. Downloaded by guest on September 29, 2021 ligands, or perhaps it induces a conformational change, possibly perturbing receptor oligomerization, which is increasingly rec- ognized as a key step in GPCR function (34). The very sharp dose–response curve observed for Prazosin (Fig. 1D) could support an oligomerization model, where a signaling cascade is triggered 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 and stability of Prazosin- and Brefeldin-induced endosome tubules are different) (Fig. 2F and SI Appendix, Fig. S4), the mechanisms of action of Prazosin and Brefeldin involve the same protein complex, 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 increased CI-M6PR localization to vesicles in addition to the Golgi. Control or DRD3 RNAi-treated cells were stained for CI-M6PR (green) and a resident Golgi marker GM130 (red). (B) Pulse and chase experiments show sorting defects in DRD3 RNAi cells. HeLa cells were treated with control or DRD3

RNAi for 3 d before Alexa 555-EGF, FITC-70 kD-dextran, and Alexa 647- BIOCHEMISTRY Transferrin were added for 10 min followed by washing out with marker- and phenol red-free medium. Pictures were taken at indicated time points in live cells. (C) Quantification of EGF and/or dextran that colocalize or partially colocalize with transferrin; (D) Endocytic cargoes are trapped in SNX1-positive endosomes after DRD3 RNAi. HeLa cells were treated with control or DRD3 RNAi for 3 d before Alexa 555-EGF (red) was added for 10 min followed by washing out with marker-free medium for indicated time points. Cells were than washed, fixed, and stained with anti-SNX1 (green). EGF that colocalizes or partially colocalizes with SNX1 was quantified. For each condition, around 50 cells were counted. Mean values from two independent Fig. 5. The COPI complex interacts with DRD3 in the presence of Prazosin experiments are shown. (Scale bar: 10 μm.) (E) Flow cytometry analysis and is involved in its effects on endocytosis. (A) Pull-down experiments using of transferrin recycling in control or DRD3 RNAi knockdown HeLa cells. anti-FLAG antibody in GFP-DRD3-FLAG-HeLa cells treated with DMSO, Pra- zosin, or Terazosin show that the interaction of DRD3 with COPI subunits COPB, COPC, and COPG is increased after Prazosin treatment. (B) COPB, and COPI both function in sorting and the addition of Prazosin COPD, and COPG localizations are disrupted by Prazosin treatment. HeLa stabilizes a nonproductive complex between them and probably, cells were treated with DMSO or 30 μM Prazosin for 1 h before fixing and other proteins (Fig. 5A). The concept of a small-molecule sta- staining with COP antibodies. (C) COPB1 or COPB2 RNAi prevents endosomal bilizing interactions between proteins is familiar from chemical tubule formation in Prazosin-treated HeLa cells. Transferrin receptor stain- inducers of dimerization (33), suggesting that Prazosin may ing is shown. HeLa cells were treated with siRNAs for 3 d before DMSO or 30 μ act similarly. M Prazosin was added for 1 h. (D) Pulse and chase experiments show Although our data suggest that the third cytoplasmic loop of sorting defects in COPB RNAi cells. HeLa cells were treated with control or COPB1 + COPB2 RNAi for 3 d before Alexa 488-Tf and FITC-70 kD-dextran D3 might be involved, exactly where Prazosin binds in this were added for 10 min followed by washing out with marker- and phenol complex is not yet clear. The fact that it antagonizes DRD1 and red-free medium. Individual COPB1 or COPB2 RNAi resulted in similar phe- -2 suggests that it can bind to closely related GPCRs and may, notypes. Pictures were taken at indicated time points without fixing the therefore, bind to DRD3 directly. Perhaps it binds to DRD3 in cells. (Scale bar: 10 μm.) (E) Flow cytometry analysis of transferrin recycling in a nonstandard manner that is not competitive with traditional control or COPB1 or COPB2 RNAi knockdown HeLa cells.

Zhang et al. PNAS | July 31, 2012 | vol. 109 | no. 31 | 12489 Downloaded by guest on September 29, 2021 Golgi but also to small vesicles and the cytoplasm. Similarly, and 1% (vol/vol) penicillin/streptomycin (P/S; Cellgro). Prazosin (P7791) and DRD3 is also found in different cellular pools (Fig. 3E). Prazosin Terazosin (T4680) are from Sigma. seems to target specific subpools of both proteins. It triggers an For all immunofluorescence experiments, cells were fixed in either 3.7% increased association between DRD3 and COPI and their absence (vol/vol) formaldehyde in PBS for 20 min or −20 °C methanol for 5 min, from the newly formed endosomal tubules. Taken together, these permeabilized with 0.1% Triton X-100 in TBS, blocked in AbDil (0.1% Triton + + data suggest that a transient but important regulatory interaction X-100 in TBS 2% (wt/vol) BSA 0.1% NaN3), and probed with primary and between DRD3 and COPI, stabilized by Prazosin, is key in regu- secondary antibodies diluted in AbDil. Cells were stained with DAPI and lating the equilibrium between vesicular and tubular endosomes mounted in Prolong gold mounting medium (Invitrogen). and therefore, endosomal sorting. The following antibodies and markers were used in immunofluorescence The effect of Prazosin on endosome dynamics is interesting and Western blots: anti-GFP, Transferrin receptor, LAMP1, Clathrin, CI-M6PR, clinically, both because endosomal trafficking is involved in nu- GM130, Giantin, COPB and COPD antibodies (Abcam), EEA1, SNX1 and SNX2 merous disease-related processes and because the action of antibodies (BD Bioscience), TRAPα antibody (a gift from Tom Rapoport, Prazosin implicates a GPCR from an important family in a basic Harvard Medical School, Boston, MA), DRD3 antibody (Calbiochem), fluo- and general biological regulatory process. Given that many rescently labeled transferrin and EGF (Invitrogen), FITC-70 kD dextran and clinically approved drugs targets GPCRs, a better understanding mouse anti-FLAG antibody (Sigma), and COPG antibody (Santa Cruz). of these receptors’ biological functions will be critical in de- fi veloping more efficacious therapeutics. Prazosin is the only Statistics. For quanti cations in the manuscript, mean values are shown in the fi fi clinically approved drug that inhibits a specific step during en- gures, and SDs are shown as error bars. All images shown in gures are docytosis, and we know from its long history that it is relatively representative. 500 (Fig. 1C), 300 (Figs. 2C,3C and D, and 5C), or 50 (Figs. 2G nontoxic. Targeting endocytic processes clinically would require and 4C) cells were counted for each condition in three independent experiments. Comparisons between treatments were analyzed by a two- higher Prazosin doses than those doses currently used in anti- fi hypertension therapy, because concentrations in patients’ blood tailed Student t test. P values are labeled in the gures for where data were compared. In Figs 2H,4E, and 5E, 10,000 cells each were collected in three are about 100-fold less than we reported here; however, Prazosin fi independent experiments. Mean fluorescence intensities normalized to can be a starting point in additional development. Our ndings 0 min are shown. highlight the increasingly accepted concept that even the most widely used and safe drugs often have unexpected targets and ACKNOWLEDGMENTS. We thank the Nikon Imaging Center at Harvard mechanisms. Off-target effects of compounds that have been Medical School for assistance with microscopy, the Flow Cytometry facility at thoroughly vetted in humans are always of great interest to both the Dana-Farber Cancer Institute for assistance with flow cytometry, and understand their efficacy and toxicity in their current indications Qingsong Liu and members of the U.S.E. laboratory for helpful discussions. and spark ideas for new indications. We also thank Maria F. Sassano and Bryan L. Roth at the University of North Carolina and the National Institute of Mental Health Psychoactive Drug Materials and Methods Screening Program for conducting the experiments shown in SI Appendix, Fig. S6. M.L.C. and T.J.M. were supported by National Institutes of Health Cell Culture and Immunofluorescence. HeLa cells were grown in monolayers in Grant R01 GM023928. This project was funded by National Institutes of DMEM supplemented with 10% (vol/vol) heat-inactivated FBS (Invitrogen) Health Grant R01 GM082834 (to U.S.E.) and the Dana-Farber Cancer Institute.

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