Dopamine Receptor D3 Regulates Endocytic Sorting by a Prazosin-Sensitive Interaction with the Coatomer COPI
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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 coat- omer 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 per- turbation 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).