Arrestin Expression in E. coli and Purification

UNIT 2.11

Sergey A. Vishnivetskiy,1 Xuanzhi Zhan,1 Qiuyan Chen,1 Tina M. Iverson,1 and Vsevolod V. Gurevich1 1

Department of Pharmacology, Vanderbilt University, Nashville, Tennessee

Purified arrestin proteins are necessary for biochemical, biophysical, and crystallographic studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in E. coli and purification of the tag-free wild-type and mutant arrestins. The method includes ammonium sulfate precipitation of arrestins from cell lysates, followed by heparin-Sepharose chromatography. Depending on the arrestin type and/or mutations, the next step is Q-Sepharose or SP-Sepharose chromatography. In many cases the nonbinding column is used as a filter to bind contaminants without retaining arrestin. In some cases both chromatographic steps must be performed sequentially to achieve high purity. Purified arrestins can be concentrated up to 10 mg/ml, remain fully functional, and withstand several cycles of freezing and thawing, provided that overall salt concentration is maintained at or above physiological C 2014 by John Wiley & Sons, Inc. levels.  Keywords: arrestin r recombinant r expression r purification r chromatography

How to cite this article: Curr. Protoc. Pharmacol. 67:2.11.1-2.11.19. doi: 10.1002/0471141755.ph0211s67

INTRODUCTION Arrestins are multifunctional adaptor proteins that form complexes with hundreds of G protein-coupled receptors (GPCRs) and dozens of other partners (Gurevich and Gurevich, 2006b, 2014; DeWire et al., 2007), and, as such, they are important in cell signaling. The mammalian arrestin family contains four members, with two visual subtypes, arrestin-1, also known as S-antigen, 48 kDa protein, visual or rod arrestin, and arrestin-4, which is sometimes referred to as cone or X-arrestin. For some reason its gene is designated as arrestin 3 in the HUGO database. The other two members of this family are ubiquitously expressed nonvisual subtypes. These include arrestin-2, also referred to as β-arrestin or β-arrestin1, and arrestin-3, also known as β-arrestin2 or hTHY-ARRX (see Gurevich and Gurevich, 2006a). The systematic names of arrestin proteins are used in this unit, with the order of cloning indicated after dash. The sequential chromatographic protocol of arrestin purification after ammonium sulfate precipitation described below is based on original protocols (Lohse et al., 1992; S¨ohlemann et al., 1995) that were modified to purify different arrestin isoforms of bovine origin (Gurevich et al., 1999; Gurevich and Benovic, 2000). We have used this protocol to purify all four bovine wild-type (WT) arrestins, arrestins from other species, and numerous mutants. The methods in the Basic Protocol have worked well for some arrestin isoforms and their variants, e.g., arrestin-2, although certain modifications in the chromatographic steps are necessary to obtain pure fully functional arrestin proteins from different species and their mutant forms. The Alternate Protocol gives procedures

Current Protocols in Pharmacology 2.11.1-2.11.19, December 2014 Published online December 2014 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471141755.ph0211s67 C 2014 John Wiley & Sons, Inc. Copyright 

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for the purification of more demanding proteins with lower expression, e.g., arrestin-3 and some mutants. The Support Protocol describes small-scale methods for testing the feasibility of large-scale expression and purification of arrestins of interest in E. coli.

STRATEGIC PLANNING Before undertaking large-scale arrestin expression for purification, it is necessary to ascertain that the protein of interest, be it a WT form from another species or a particular bovine mutant, is expressed at sufficiently high levels. The Support Protocol describes test expression in E. coli, performed along with a well expressed arrestin protein as a positive control to determine whether large-scale expression and purification of that target arrestin is feasible. In our experience, nine out of ten WT or mutant arrestins are expressed at levels high enough to make large-scale purification possible. Usually the higher the expression level, the higher the overall yield and purity of the final sample. Generally, for large-scale purification, the expression of soluble arrestin must be greater than 1 mg per liter of bacterial culture. Arrestins are purified using Sepharose columns, first with heparin-Sepharose and then with Q-Sepharose and/or SP-Sepharose. In some cases, an additional SP-Sepharose column is used to improve the purity of arrestin proteins (Gurevich et al., 1999; Gurevich and Benovic, 2000). Arrestin proteins display different chromatographic profiles on Q- and SP-Sepharose, which are anion and cation exchangers, respectively. Figure 2.11.1 summarizes two different strategies for chromatographic purification. The first, strategy (A), is designed for arrestin proteins that bind weakly to both Q- and SP-Sepharose columns, e.g., bovine visual arrestin-1. In the process of loading the samples onto Q-Sepharose, the eluate from heparin-Sepharose is diluted to a low salt concentration (95% purity.

BASIC PROTOCOL

Materials LB broth (Sigma): prepare according to the manufacturer’s instructions 100 mg/ml ampicillin (Amp) stock solution in distilled water: store at 4o C Cells transformed with arrestin expression constructs, glycerol stock (see Gurevich et al., 1999; Gurevich and Benovic, 2000): available from V.V. Gurevich upon request ([email protected]) 100 mM isopropyl-β-D-thiogalactopyranoside (IPTG): prepare in distilled water; store up to 2 years at −80°C Lysis buffer (see recipe) Lysozyme from chicken egg white (Sigma) 1.0 M magnesium chloride solution (Sigma) 15,000 U deoxyribonuclease I from bovine pancreas (DNase I; Sigma)/ml lysis buffer (see recipe) 20,000 U deoxyribonuclease II from porcine spleen (DNase II; Sigma)/ml lysis buffer (see recipe) 100 mg ribonuclease A from bovine pancreas (RNase A; Sigma)/ml lysis buffer (see recipe) Ammonium sulfate Multiple column buffer (MCB; see recipe) Heparin-Sepharose 6 Fast Flow (GE Healthcare) 100 mM, 150 mM, 200 mM, 2 M, and 3 M NaCl in MCB (see recipe) 100% (v/v) methanol 2× SDS sample buffer (Sigma) 10% (w/v) SDS-PAGE gel Q-Sepharose Fast Flow (GE Healthcare)

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SP-Sepharose Fast Flow (GE Healthcare) F431 rabbit polyclonal antibody (see Song et al., 2011): available from V.V. Gurevich upon request ([email protected]) 500-ml flasks 2-liter flasks 30°C incubator with shaking platform RC-5B Plus centrifuge with SLA 3000 rotor (Sorvall) 500-ml centrifuge bottles Sonicator (Fisher Dimembrator model 500, or equivalent) TL-100 tabletop ultracentrifuge with TLA 120.1 rotor (Beckman) 1.5-ml microcentrifuge tubes 1-liter beaker Orbital shaker 0.8-μm syringe filter, sterile (Corning) 1.5 × 20–cm Econo-Column chromatography columns (Bio-Rad, cat. no. 737-1522) GradiFrac chromatography system (Pharmacia Biotech) Amicon filter unit with YM30 filters (Millipore) Additional reagents and equipment for carrying out SDS-PAGE electrophoresis (APPENDIX 3B; Gallagher and Sasse, 1998), western blotting (Gallagher et al., 2011; also see Gurevich et al., 1999; Gurevich and Benovic, 2000), and protein quantification (APPENDIX 3A; Olson and Markwell, 2007) Grow cells and express arrestin 1. Prepare one sterile 500-ml flask with 160 ml of LB broth and four 2-liter flasks with 1.1 liter LB each. 2. Add 160 μl of the ampicillin stock solution to 160 ml LB (LB/Amp). 3. Inoculate this LB/Amp with selected arrestin/BL21 cells from the glycerol stock. With a pipet tip, scrape 30 to 60 μl off the frozen cell suspension. Grow the cells to OD600 of 0.1 to 0.2 for 6 to 8 hr at 30°C, with continuous shaking at 250 rpm. 4. Add 40 ml of this culture to each flask with 1.1 liter LB/Amp. Grow overnight to OD600 of 1.0 to 1.5. 5. Add 275 μl (or 385 μl for arrestin-3) of IPTG stock solution to each flask to a final concentration of 25 μM or 35 μM, respectively, to induce arrestin expression. Continue shaking for an additional 4 to 5 hr at 30°C, 250 rpm.

Lyse cells 6. Centrifuge the cells 10 min at 6000 × g, 4°C (SLA 3000 rotor RC-5B Plus) in six 500-ml centrifuge bottles. Two rounds of centrifugation are required to collect cells from 4.5 liters of culture. All of the bottles should contain roughly equal amounts of cells.

7. Carefully discard the supernatant, and stand the tubes upside down for 1 min, wiping away all traces of supernatant with Kimwipes. NOTE: All subsequent steps are performed on ice or in a cold room.

Arrestin Expression in E. coli and Purification

8. Add 22 ml lysis buffer to each tube, and thoroughly resuspend the cells by pipetting. Avoid frothing. Measure the total volume of the cell suspension in each bottle. 9. Add 1 ml fresh 3 mg/ml lysozyme dissolved in lysis buffer to each bottle, and incubate 30 to 40 min on ice.

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10a. To store the cells: Freeze the cell lysate at −80°C, and store up to several weeks at −80°C. 10b. To proceed immediately: Freeze the cell lysate 20 min at −80°C, and then thaw on ice for 30 to 60 min. 11. Add 80 ml of ice-cold lysis buffer to each bottle to a final volume of 100 ml per bottle. Carefully mix to ensure that the frozen cell suspension is fully thawed. 12. In an ice-water bath, sonicate the cell lysate three times (15 sec each time, 90% amplitude), with 1 to 2 min intervals between rounds to allow the suspension to cool. 13. On ice, add 0.88 ml of 1 M MgCl2 to a final concentration of 7 to 8 mM (to ensure that free Mg2+ is present), 160 μl of 15,000 U/ml DNase I , 160 μl of 20,000 U/ml DNase II , and 160 μl of 100 mg/ml RNase A to each bottle. Mix the samples thoroughly, and incubate 40 min on ice. As an option, a 100-μl aliquot of the homogenate can be transferred to a microcentrifuge tube (labeled homogenate sample), and stored up to several weeks at −20°C. This sample can be compared with supernatant (see step 15) by western blot to determine the fraction of soluble arrestin.

14. Centrifuge the homogenate 90 min at 13,000 × g, 4°C. 15. Carefully collect the supernatant into a clean, ice-cold 1-liter beaker. Determine the volume, which usually equals the total volume of added lysis buffer, or 600 ml. Remove a 100-μl aliquot from the supernatant, place it in a microcentrifuge tube (labeled supernatant sample), and store up to several weeks at −20°C.

Carry out ammonium sulfate precipitation 16. In a 4°C cold room, add 192 g (0.32 g/ml, final concentration 2.4 M) of ammonium sulfate, in four portions (25% at a time) to the 600 ml of the supernatant, stirring gently to avoid frothing. Allow the stirring to continue until the ammonium sulfate is completely dissolved. Do not add the ammonium sulfate all at one time, as this will cause the initial concentration to be much higher than necessary. Avoid frothing, as proteins denature at the water-air interface.

17. Collect the precipitated protein in two 500-ml bottles by centrifuging 90 min at 20,500 × g, 4°C (SLA 3000 rotor, RC-5B Plus centrifuge). Carefully remove the supernatant and floating white material. These pellets can be stored in covered bottles at −80°C until needed. They are stable for at least a month.

Prepare samples for heparin-Sepharose chromatography All procedures related to the chromatography are performed at 4°C. 18. To prepare the heparin-Sepharose loading sample, dissolve the protein precipitated by ammonium sulfate in 200 ml of ice-cold MCB, making certain the protein pellet is completely dissolved by gently shaking on an orbital shaker. Here, and at each step below, add phenylmethanesulfonyl fluoride (PMSF) to the MCB just before use. Starting from this point, all further steps are performed on ice or in the cold room.

19. To remove the insoluble material, centrifuge the protein solution in a 500-ml bottle 90 min at 20,500 × g, 4°C (SLA 3000 rotor, RC-5B Plus centrifuge).

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Table 2.11.1 Heparin-Sepharose Loading and Elution Conditions

Arrestins

Loading conditions

Elution conditions

Bovine arrestin-1

100 mM NaCl

Elution gradient: MCB/100 mM NaCl—> MCB/500 mM NaCl Arrestin peak: 190-270 mM NaCl

Bovine arrestin-2

150 mM NaCl

Elution gradient: MCB/150 mM NaCl—> MCB/0.8 M NaCl Arrestin peak: 442-523mM NaCl

Bovine arrestin-3

200 mM NaCl

Elution gradient: MCB/200 mM NaCl—> MCB/1 M NaCl Arrestin peak: 380-500 mM NaCl (full-length) 633-703 mM NaCl (truncated)

20. Carefully collect the supernatant, while avoiding the pellet, and filter it through an 0.8-μm sterile syringe filter. Determine the volume of the supernatant to estimate the volume of the initial pellet. This is usually 15 to 20 ml.

21. Place a 100-μl aliquot of the supernatant into a microcentrifuge tube (labeled heparin-input sample), and freeze at −20°C. 22. Dilute the sample with MCB to the appropriate salt concentration before loading it onto the heparin-Sepharose column (Table 2.11.1). Generally, the salt concentration of the sample during loading should be 30% to 45% of that of the elution peak. An example of how to calculate the sample dilution involves the use of arrestin-3. In this case, as the arrestin-3 is eluted from heparin-Sepharose at 380 to 500 mM NaCl, the salt concentration of the loading sample is set at 200 mM NaCl. The pellet contains 2.4 M ammonium sulfate, which is equivalent to 7.2 M NaCl solution in terms of ionic strength. Therefore, for a 20-ml pellet, the final volume of the loading sample should be 720 ml (36-fold dilution relative to pellet).

Load and wash the heparin-Sepharose column 23. Under gravity flow, pack a 25-ml column (1.5 × 20–cm with heparin-Sepharose 6. Wash this column first with 3 M NaCl and then with 200 ml of 200 mM NaCl/MCB (100 mM for arrestin-1, 150 mM for arrestin-2, and 200 mM for arrestin-3). 24. Load the protein sample onto the column, and collect the flow-through for further analysis. Because the MCB has a very low ionic strength, its addition to the filtrate sometimes results in precipitation of protein, thus clogging the column. Therefore, it is advisable to dilute the sample during column loading using a gradient mixer and an appropriate ratio of filtrate and MCB. Because of the large volume, loading is usually performed overnight at a flow rate that ensures that the column does not dry out by morning.

25. The next morning, wash the heparin-Sepharose column with 100 ml of 200 mM NaCl/MCB. Collect the wash for further analysis.

Arrestin Expression in E. coli and Purification

26. Elute proteins from the heparin-Sepharose column with a 400-ml linear gradient of NaCl and collect forty 10-ml fractions. See Table 2.11.1 for the elution conditions for some arrestin proteins. 27. After the gradient is completed, wash the column with 50 ml of 3 M NaCl/MCB (high salt elution), collecting 10-ml fractions.

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full-length arrestin-3 (elution pool) M

S

PT

W

5

7

9

10

11

12

13

14 15

16

17

200 150 100 75 arrestin-3

50 37

25 20

18

M 19

20

21

22 23

24 25

26

28

32

36

40 2M NaCl

full-length truncated

Figure 2.11.2 Coomassie-stained gels of arrestin-3 fractions eluted from heparin-Sepharose. Fraction numbers and positions of full-length and in vivo truncated arrestin-3 are shown. Molecular masses of markers (M) are given on the left of the top gel. PT, pass-through; S, supernatant (sample loaded onto column); W, wash.

Identify arrestin-containing fractions by electrophoresis and western blot 28. Remove 30-μl aliquots from each 10-ml fraction, and transfer them to clean 1.5-ml microcentrifuge tubes, along with aliquots of the input, flow-through, and wash. For these samples the aliquots should equal 1:100,000 of the total volume.

29. To precipitate the protein, add 270 μl methanol (90% final), and centrifuge the sample 10 min at 16,200 × g, room temperature. 30. Carefully decant the supernatant and add 1 ml of 90% methanol to the pellets. Vortex the samples thoroughly, and centrifuge 10 min at 16,200 × g, room temperature. 31. Discard the supernatant and air-dry the samples for 10 to 15 min. The tubes should not have any methanol residue.

32. Add 15 to 20 μl of the 2× SDS sample buffer to each tube, thoroughly dissolve the pellet by vortexing, load it onto a 10% SDS-PAGE gel, and electrophorese. Visualize the proteins by Coomassie blue staining (Fig. 2.11.2).

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Coomassie

western blot M S PT W 5 7 9 11 12 13 14 15 16 17 18

M S PT W 5 7 9 11 12 13 14 15 16 17 18

(1-378)

(1-378)

(1-378) (1-378)

19 M 20 21 22 23 24 25 26 28 30 32 34 36 42

19 M 20 21 22 23 24 25 26 28 30 32 34 36 42

Figure 2.11.3 Elution of enhanced truncated Cys-less arrestin-1 eVSA-QH-Tr (C63V, C128S, C143A, K257Q, E346H) and 1-378, the bovine Cys-less arrestin-1 analog of the most potent in terms of binding to unphosphorylated, light-activated rhodopsin mouse mutant (Vishnivetskiy et al., 2013), from heparin-Sepharose. Coomassiestained gels (left panels) do not allow unambiguous identification of fractions to pool, making it necessary to rely on western blots (right panels). Fraction numbers are shown, with numbers greater than 40 indicating high-salt wash. M, molecular weight markers; PT, pass-through; S, protein precipitated by ammonium sulfate and then dissolved and loaded onto heparin-Sepharose; W, wash.

When purifying a new arrestin variant, use 90% of the sample for Coomassie gel and 10% for western blot with anti-arrestin antibodies (step 33). This is also important when the overall expression level is not very high, e.g., with truncated Cys-less enhanced arrestin-1 (Vishnivetskiy et al., 2013; eVSA-QH-Tr, Fig. 2.11.3). This is because it is challenging to identify the arrestin band on the Coomassie-stained gel of the heparin-Sepharose fractions, making it difficult to decide which fractions should be pooled for the next step, without parallel western analysis (Fig. 2.11.3).

33. Perform a western blot with primary F431 rabbit polyclonal antibody. F4C1 is a mouse monoclonal antibody that detects the epitope DGVVLVD, a sequence present in the all known mammalian arrestins (Donoso et al., 1990). F431is a rabbit polyclonal antibody that detects the same epitope (Song et al., 2011). Whereas the cells producing monoclonal F4C1 antibody have been lost, rabbit polyclonal F431 antibody is available from Dr. V.V. Gurevich upon request. Some commercial antiarrestin antibodies might be suitable for the task, but the authors have not tested them. We will gladly provide upon request aliquots of purified arrestin proteins to those willing to test commercial antibodies to compare them with the F431 employed by our group.

Arrestin Expression in E. coli and Purification

34. Carefully pool the fractions according to the gels, and estimate the final salt concentration in the combined fractions. For visual arrestin-1 and many of its mutants, and for arrestin-2, pool the peak arrestin-containing fractions eluted from the heparinSepharose. To ensure that only full-length arrestin-3 is purified, carefully pool the fractions containing this form at 380 to 500 mM NaCl (see Table 2.11.2).

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Table 2.11.2 Purification of WT and Mutant Arrestins from Different Species

Elution peak (mM NaCl) Organism

Arrestin

Protein

Bos taurus

Arrestin-1 WT

Heparin- Q-Sepharosea SPYield (mg/liter Sepharose Sepharosea,b bacterial culture 190-270

54-80

+

4-5

350-450

12-42

−

5

34-100

+

8

442-523

97-164

−

13.5

3A

596-681

150-200

−

3.9

1-382

623-707

27-80

176-225

7.8

120-165

92-145

10

380-500

+

227-327

3.4

633-703

17-43

−

0.6

10-55

50-130

−

1.35

170-300

120-226

−

9.85

183-289

18-63

+

2.8

Arrestin-4 WT

10-55

40-130

−

0.4

Arrestin-1 WT

114-341

105-142

−

8.6

450-590

182-208

−

0.44

90-140

142-165

−

1.4

1-378

Cys-less 210-290 Arrestin-2 WT

Cys-less 426-554 Arrestin-3 WT 1-382 Mus musculus Arrestin-1 WT 3A Homo sapiens Arrestin-1 WT Ambystoma tigrinum

3A Arrestin-4 WT a The b The

+ symbol indicates the column is used as a filter to bind impurities and allow the arrestin flow-through. − symbol indicates the column is not used.

Although most arrestins are not subject to in vivo proteolysis in E. coli, the C-tail of arrestin-3 is often cleaved by proteases. Although different kinds of protease inhibitors may be added, in our experience up to half of the arrestin-3 is truncated (cleaved at residue 390). The full-length and truncated forms are partially separated on heparinSepharose (Fig. 2.11.2).

35a. To further purify arrestin-3 and the truncated form of arrestin-1-(1-378): Go to the Alternate Protocol. 35b. To further purify arrestin-1 and arrestin-2: Proceed with the following steps.

Purify arrestin-1 and arrestin-2 fractions by Q-Sepharose and SP-Sepharose 36. Combine the fractions containing the peak of arrestin, concentrate them in an Amicon filter unit to 10 ml, and then filter the sample through a 0.8-μm filter. 37. Transfer a 100-μl aliquot of the supernatant to a 1.5-ml microcentrifuge tube (labeled heparin sample), and freeze at −20°C. 38. Pack under gravity flow 10 ml of Q-Sepharose and SP-Sepharose in 1.5 × 20–cm columns, and wash them with 200 ml MCB at 4o C. 39. Estimate the concentration of NaCl in the concentrated pooled fractions based on the gradient used, taking into account that the salt concentration after 25 ml of the column is delayed by three fractions. For example, in the case of arrestin-1, the gradient is 100 mM to 500 mM. Thus, the salt content in each fraction is higher than in the previous one by 10 mM, so NaCl in fraction 15 should be around 100 mM + (15 – 3) × 10 mM, which is 220 mM.

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heparin-Sepharose

Q-Sepharose

M S PT W 5 7 9 1112 13 14 15 16 17 18

M H PT 2 3 4 5 6 7 8 9 10 11 12 13

arrestin

arrestin

19 M 20 21 22 23 24 25 27 29 31 33 35 37 42

14 15 M 16 17 18 19 20 21 22 23 25 27 29 32

Figure 2.11.4 Two-step purification of Cys-less arrestin-1 eNCL (C63V, C128S, C143V; Hanson et al., 2006b; Vishnivetskiy et al., 2010). Coomassie-stained gels for heparin-Sepharose (left panels) and subsequent QSepharose (right panels) are displayed. Fraction numbers are shown, with numbers greater than 40 after heparin and greater than 30 after Q indicating high-salt wash. H, pooled fractions from heparin-Sepharose; M, molecular weight markers; PT, pass-through; S, protein precipitated by ammonium sulfate and then dissolved and loaded onto heparin-Sepharose; W, wash.

40. Load the sample onto a 10-ml Q-Sepharose column while diluting it with 0.2× MCB (dilute MCB buffer 5-fold) using a gradient mixer to a salt concentration that is 95%) purity. Skip the SP-Sepharose column step for these proteins.

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Further purify arestin-1 by SP-Sepharose chromatography 44. Load pooled fractions onto a 10-ml SP-Sepharose column, and collect the flowthrough. At 100 mM NaCl, visual arrestin-1 directly flows through the SP-Sepharose, while some contaminants bind.

45. Concentrate the SP-Sepharose flow-through containing visual arrestin-1 (>95% purity) to 3 to 5 mg/ml in an Amicon filter unit. To eliminate aggregates formed during the concentration step, filter the concentrated sample through a 0.8-μm filter. 46. Measure the protein concentration (APPENDIX 3A; Olson and Markwell, 2007). 47. Dispense aliquots of arrestin samples, and store up to 2 years at −80°C. Freezing arrestins does not appreciably reduce their activity for at least 2 years, with up to two to three freeze-thaw cycles during this time.

PURIFICATION OF ARRESTIN-3 AND TRUNCATED FORM OF ARRESTIN-1-(1-378)

ALTERNATE PROTOCOL

After step 34 of the Basic Protocol, use the following procedure to purify arrestin-3 and truncated arrestin-1(1-378). Arrestin-3, as well as truncated arrestin-1-(1-378), bind to SP-Sepharose relatively tightly. To avoid a time-consuming concentration step, use Q-Sepharose as a filter column before binding arrestin proteins to SP-Sepharose.

Additional Materials (also see Basic Protocol) Heparin-Sepharose arrestin fractions (from Basic Protocol, step 34) 5.0 M NaCl solution (Sigma) 1. Pool heparin-Sepharose fractions, and estimate the NaCl concentration. When necessary (see Table 2.11.2), add 5 M NaCl to a final concentration of 100 mM. 2. Transfer a 100-μl aliquot of the supernatant to a microcentrifuge tube (labeled heparin sample), and store up to several weeks at −20°C. 3. Pack under gravity flow 10 ml Q-Sepharose and SP-Sepharose in 1.5 × 20–cm columns, and wash them with 200 ml of MCB at 4°C. 4. Sequentially connect the Q- and SP-Sepharose columns (Fig. 2.11.1B). Load the samples onto the Q-Sepharose column. At 100 mM NaCl, arrestins flow through Q-Sepharose, binding only to SP-Sepharose.

5. Wash the Q- and SP-Sepharose columns with 50 ml of 100 mM NaCl/MCB. 6. Disconnect the SP-Sepharose from the Q-Sepharose. Elute arrestin from the SPSepharose column with a 300-ml linear gradient of NaCl (100 mM NaCl/MCB to 500 mM NaCl/MCB). Collect thirty 10-ml fractions. 7. After the gradient elution, wash the column with 50 ml of 2 M NaCl/MCB (high salt elution) and collect 10-ml fractions. Analyze the fractions by SDS-PAGE, as described in the Basic Protocol. 8. Pool the arrestin-containing fractions, and estimate the final NaCl concentration. Concentrate the fractions to 3 to 5 ml using an Amicon filter unit. To eliminate aggregates formed during the concentration step, filter the concentrated sample through an 0.8-μm filter. 9. Measure the protein concentration (APPENDIX 3A; Olson and Markwell, 2007).

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M

lysate

sup

heparin

SP

200 150 100 75

50

37

25

Figure 2.11.5 Purity of arrestin-3 through purification steps. The positions of molecular weight markers (M) and protein content of lysate, supernatant after dissolution of ammonium sulfateprecipitated protein (sup), pooled heparin-Sepharose (heparin), and SP-Sepharose (SP) fractions are displayed. Table 2.11.3 Typical Yields of Total Protein at Sequential Steps of Purificationa

Protein (mg) Purification step

Bovine arrestin-1

Bovine arrestin-2

Bovine arrestin-3

Homogenate

2,200

2,600

2,900

Supernatant

1,100

1,500

1,600

580

100

30

20

50

–

–

–

13

Heparin-Sepharose b

Q-Sepharose

b

SP-Sepharose

are achieved by a moderately experienced person; yields can vary ± 50%. protein resulting from this purification step is >95% pure.

a These b The

10. Dispense aliquots of arrestin samples, and store up to 2 years at −80°C. Figure 2.11.5 shows typical arrestin-3 purities at different steps of purification, and Table 2.11.3 shows the usual protein yields at each step. The final number is the yield of purified arrestin. SUPPORT PROTOCOL

Arrestin Expression in E. coli and Purification

SMALL-SCALE TEST EXPRESSION OF WILD-TYPE AND MUTANT ARRESTINS IN E. coli The aim of the protocol described in this unit is to determine the relative expression level of different arrestins in E. coli. Purification of any protein is normally a time-consuming process. In the case of arrestin, 5 to 6 days are needed to obtain pure, high-quality protein. Not all arrestins behave the same in the E. coli expression system. For example, bovine arrestin-2 is expressed at levels twice those of bovine arrestin-1, whereas bovine arrestin-3 is expressed at lower levels than either arrestin-1 or -2. Sometimes substitution

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e35C

e35-173C

e60-173C

eNCL

supernatant

e35C

e35-173C

e60-173C

eNCL

homogenate

e35-173CTr

e60-173CTr

e12-173CTr

eNCL

supernatant

e35-173CTr

e60-173CTr

e12-173CTr

eNCL

homogenate

Figure 2.11.6 Western blots of test-expression samples of truncated (1-378; left panel) and fulllength (right panel) bovine arrestin-1 mutants of Cys-less VSA (C63V, C128S, C143A) background with cysteines in the indicated positions, compared with eNCL (C63V, C128S, C143V). The blots of supernatant are much more informative than those of homogenate. Note several-fold differences in expression levels, although all proteins are expressed sufficiently to perform a large-scale expression and purification.

of a single amino acid in bovine arrestin-1 can dramatically change its behavior. In our experience, nine out of ten mutants are expressed well, whereas some are not expressed at a level sufficient for purification. Test expression of the desired arrestins using either the well characterized bovine arrestin-1 or another highly expressing species as a positive control in a small volume of E. coli cells provides an estimate of what to expect and whether large-scale purification should be attempted. As an example, the results of the test expression of the bovine Cys-less arrestin-1 and its full-length and truncated mutants with single or double cysteine substitutions are shown (Fig. 2.11.6).

Additional Materials (also see Basic Protocol) 15-ml conical, plastic tubes, sterile 500-μl centrifuge tube (Beckman) Grow cells and express arrestin 1. Prepare one sterile, conical, plastic 15-ml tube with 3 ml LB broth for each arrestin protein to be test-expressed, including the positive control. Add 3 μl of the ampicillin stock solution into each tube. 2. Inoculate each tube with 20 to 30 μl of frozen arrestin/BL21 cells that have been scraped with a pipet tip off the unthawed glycerol stock. Allow the cells to grow overnight (12 to 16 hr) at 30°C, with continuous shaking at 250 rpm. 3. Add 7.5 μl or 10.5 μl (for arrestin-3) of 0.01 M IPTG to each tube to final concentrations of 25 μM and 35 μM, respectively, to induce the expression of arrestin. Continue incubating at for an additional 3 to 4 hr at 30°C, with shaking at 250 rpm. 4. Centrifuge the cells 10 min at 6000 × g, 4°C. 5. Carefully discard the supernatant, and then allow the tubes to stand for 1 min upside down to remove all traces of the LB broth. When performing test-expression of several proteins, prepare solutions sufficient for an extra sample. For example, when expressing nine proteins, prepare solutions for ten to allow for loss and pipetting errors.

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Lyse cells 6. Add the protease inhibitor and dithiothreitol (DTT) to the lysis buffer in appropriate volumes (e.g., 2 μl of 1.0 M PMSF and 1 μl of 4.0 M DTT to 200 μl of lysis buffer). Note that no protease inhibitor cocktail is added to lysis buffer for the following steps. The final concentration of DTT is normally 2 mM, but it is increased to 5 mM with single Cys mutants on Cys-less background to prevent oxidation of those Cys. DTT may be added to a volume of lysis buffer sufficient for all of the following steps, provided they are carried out within 1 to 2 hr. The PMSF must be added immediately before use, as its half-life in water is minutes. Starting from this point, perform all remaining steps on ice. Here and at each step below add PMSF to the lysis buffer immediately before use.

7. Add the 200 μl of ice-cold lysis buffer to the bacterial pellet, and resuspend the cells by vortexing. Transfer the cell suspension to a 1.5-ml microcentrifuge tube. 8. Add the 200 μl of ice-cold lysis buffer with 0.1 mg/ml lysozyme to the 200 μl of the cell suspension and vortex. Maintain on ice for 40 min. 9. Freeze the cells at −80°C 20 min or until fully frozen. Then allow the cells to thaw on ice. The freeze-thaw cycle helps rupture the bacterial cell membrane because the ice crystals formed upon freezing cause the bacteria to swell.

10. Add the 200 μl of lysis buffer to the thawed cell suspension and sonicate the sample in an ice-water bath for 15 sec at 12% amplitude. 11. Add to the sample 4 μl of 1 M MgCl2 , 2 μl of 100 mg/ml RNase A, and 2 μl of 15,000 U/ml DNase I, and incubate 40 min on ice. Sonication, as well as DNase and RNase treatments, helps to shear DNA and RNA after lysis of the bacterial cells, reducing the viscosity of the lysate.

12. Transfer 200 μl to a new microcentrifuge tube (labeled homogenate), and store up to several weeks at −80◦ C. 13. Transfer the remaining 400 μl to a 500-μl centrifuge tube and centrifuge 30 min at 356,000 × g, 4o C, in aTL-100 tabletop ultracentrifuge, with aTLA 120.1 rotor. 14. Transfer the supernatant to a new microcentrifuge tube (labeled supernatant), and store up to several weeks at −80°C.

Carry out western analysis 15. To prepare samples for western analysis, remove 5 μl of the homogenate and supernatant and dissolve them in 15 μl of the 2× sample buffer. 16. Electrophorese 2 μl of each sample on a 10% SDS-PAGE gel, and carry out a western blot with F431 antibody or any other arrestin-specific antibody. 17. Compare the intensity of the arrestin band with that of a known protein, e.g., bovine arrestin-1 or arrestin-2 that is expressed in parallel.

REAGENTS AND SOLUTIONS Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX. Arrestin Expression in E. coli and Purification

Lysis buffer For 200 ml of buffer, combine the following components:

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10 ml of 1.0 M Tris·Cl buffer solution, pH 8.0 (Amresco; 50 mM final concentration) 0.4 ml of 1.0 M benzamidine stock solution in distilled water (store up to several months at −20°C; 2 mM final concentration) 0.8 ml of 0.5 M ethylene glycol tetraacetic acid solution (EGTA, BioWorld; 2 mM final concentration) 2 ml of 1.0 M glucose stock solution in distilled water (10 mM final concentration): store up to 1 year at −20°C. Immediately before use add the following: 0.1 ml of 4.0 M dithiothreitol (DTT; 2 mM final concentration): store up to 1 year at −20°C 0.2 ml of 1.0 M phenylmethanesulfonyl fluoride (PMSF; 1 mM final concentration): prepare fresh in water-free DMSO 5 ml of protease inhibitor cocktail for E. coli (Sigma, cat. no. P-8465): prepare according to manufacturer’s instructions. IMPORTANT NOTE: Add DTT and all protease inhibitors immediately before use, especially PMSF, which is unstable in aqueous solutions. Lysis buffer without DTT and protease inhibitors may be stored up to several weeks at 4°C.

Multiple column buffer (MCB) For 1 liter of buffer, combine the following components: 10 ml of 1.0 M Tris·Cl buffer solution, pH 7.5 (Amresco; 10 mM final concentration) 2 ml of 1.0 M benzamidine stock solution in distilled water (store up to several months at −20°C; 2 mM final concentration) 4 ml of 0.5 M ethylene glycol tetraacetic acid solution (EGTA, BioWorld; 2 mM final concentration) 4 ml 0.5 M ethylenediaminetetraacetic acid (EDTA, Amresco; 2 mM final concentration) Immediately before use add the following: 0.5 ml of 4.0 M dithiothreitol (DTT; 2 mM final concentration): store up to 1 year at −20°C 1 ml of 1.0 M phenylmethanesulfonyl fluoride (PMSF; 1 mM final concentration): prepare fresh in water-free DMSO IMPORTANT NOTE: Add DTT and all protease inhibitors immediately before use, especially PMSF, which is unstable in aqueous solutions. MCB without DTT and protease inhibitors may be stored up to several weeks at 4°C.

COMMENTARY Background Information Originally discovered as GPCRdesensitizing proteins (Lohse et al., 1990; Attramadal et al., 1992), nonvisual arrestins are now known to also serve as versatile regulators of GPCR signaling, sequestration, and trafficking (Goodman et al., 1996; Laporte et al., 1999; Luttrell et al., 1999; Gurevich and Gurevich, 2006a; DeWire et al., 2007). Their broad functional importance has attracted a great deal of attention and sparked studies to investigate the structural and molecular

basis of their functions. These studies have been greatly enhanced by the development of methods to purify fully functional arrestins (Lohse et al., 1992; S¨ohlemann et al., 1995; Gurevich et al., 1997). Purified arrestins are used to obtain the crystal structures of the basal (Hirsch et al., 1999; Han et al., 2001; Sutton et al., 2005; Zhan et al., 2011a) and active-like (Kim et al., 2013; Shukla et al., 2013) conformations for biophysical studies of receptor-induced conformational changes (Kim et al., 2012; Zhuang et al., 2013),

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regulation of arrestin-mediated signaling (Kook et al., 2014; Zhan et al., 2011b, 2013, 2014), oligomerization (Hanson et al., 2007b; Hanson et al., 2008; Kim et al., 2011; Song et al., 2013; Chen et al., 2014), and the binding surface of arrestins engaged by GPCRs (Hanson et al., 2006b) and other partners (Wu et al., 2006; Hanson et al., 2006a, 2007a). The protocols described here for arrestin expression in E. coli are designed to yield soluble, properly folded protein that is fully functional. After lysis, insoluble material, which includes considerable amounts of some arrestins, is pelleted along with cell debris. Partial purification, as well as elimination of DNA and RNA fragments that preclude heparin-Sepharose binding, is achieved by ammonium sulfate precipitation (Lohse et al., 1992). The first chromatographic step, heparin-Sepharose chromatography, has its basis in the relatively high-affinity binding of arrestins with heparin (Palczewski and Hargrave, 1991; Gurevich et al., 1994) and in the considerable purification that can be (S¨ohlemann et al., 1995). An anion exchange step using Q-Sepharose was also used in earlier work, although without the preceding heparin-Sepahrose step (Lohse et al., 1992). On the basis of many years of experience, we have combined previously tested methods and cation exchange on SP-Sepharose into a unified procedure. The protocol described in this unit is appropriate for the purification of all four mammalian arrestins, along with most mutants, e.g., the phosphorylation-independent truncated and 3A forms (Gurevich, 1998; Song et al., 2009), as well as the cysteine-free forms (Hanson et al., 2006b, 2007b; Vishnivetskiy et al., 2011; Kim et al., 2012; Zhuo et al., 2014). The two chromatographic procedures described originally (Gurevich et al., 1999; Gurevich and Benovic, 2000) are supplemented with an additional SP-Sepharose step, where either the Q- or SP-Sepharose column serves as a filter to remove certain impurities, while arrestin binds to the other column and is then eluted with a NaCl gradient. These modifications eliminate time-consuming and often yield-lowering concentration steps. The higher arrestin-binding capacity of SP-Sepharose compared to Q-Sepharose also dramatically increases the yields of purified arrestin proteins, especially arrestin-3, which is not expressed as readily as other subtypes and is subject to in vivo proteolysis in E. coli.

Critical Parameters The induction of arrestins by low concentrations of IPTG at 30°C is specifically optimized for maximum yields of properly folded functional arrestin in bacterial cytoplasm. Whereas a higher overall expression can be achieved at higher IPTG concentrations, in this case, most arrestins are in the insoluble fraction, probably as inclusion bodies. Because multimilligram quantities of pure arrestins are obtained, there is enough for either crystallization (Han et al., 2001; Hirsch et al., 1999; Sutton et al., 2005; Zhan et al., 2011a) or demanding biophysical studies (Hanson et al., 2006b, 2007a; Kim et al., 2011, 2012; Vishnivetskiy et al., 2011; Zhuang et al., 2013; Zhuo et al., 2014). Because they can be produced in a soluble form, there has not been an attempt to refold arrestins from inclusion bodies. Accordingly, the most critical parameter for the purification of arrestins using this protocol is the expression level of correctly folded protein, which is estimated by smallscale test-expressions (Support Protocol). It is virtually impossible to purify any wild-type or mutant arrestin that is expressed at 100 mM NaCl before ultrafiltration. In addition, concentrating most arrestins above 10 mg/ml should be avoided, as this often leads to aggregation. As arrestins do not aggregate significantly upon freezing, these proteins can survive several freeze-thaw cycles. However, when even a small fraction of aggregated protein is detrimental to the experiment, thawed arrestins should be centrifuged 1 hr at >100,000 × g, 4°C, with only the top four-fifths of the supernatant used to exclude any traces of the pellet. Modification of single-cysteine arrestins with spin labels (Hanson et al., 2006a,2006b, 2007a,2007b; Vishnivetskiy et al., 2010, 2011; Zhuo et al., 2014) or fluorescent labels (Ahmed et al., 2011) for 1 to 2 hr at room temperature often results in aggregation. These procedures can be safely performed overnight at 4°C.

Anticipated Results Expected yields Table 2.11.3 shows the expected yields of bovine wild-type arrestins at >95% purity. The yields of certain mutants and/or arrestins from different species can be much lower. A good guide is an estimate of the amount of arrestin of interest in soluble form in lowscale test-expression (Support Protocol), with 30% overall yield after purification. Thus, if the arrestin of interest yields 1 mg/liter of soluble protein, which is 4 to 5 mg per 4.5liter preparation as described here, there is a final yield of 1.5 to 2 mg of pure protein, the minimum needed for a feasible purification. Lower expression levels result in disproportionally lower yields because of losses associated with the process. Sample manipulation instructions When purifying a new protein, or any arrestin with a lower yield, it is important to analyze all chromatography fractions using both a Coomassie-stained SDS-PAGE gel and a western blot (Fig. 2.11.3). All arrestins run slowly on SDS-PAGE. Thus, although arrestin-1 is 44 kDa, it runs at 50 kDa, whereas arrestin-2 is 46 kDa, but runs above 50 kDa. Although not quantitative, western blots do indicate where the majority of the protein of interest elutes, allowing for the ready identification of the Coomassie band that corresponds to the ex-

pressed protein. Because Coomassie staining is more quantitative than western blots, the fractions should be pooled on the basis of the Coomassie-stained gels. It is best to run samples from different stages of purification sideby-side (Fig. 2.11.5), loading the same amount of total protein (1 to 2 μg) in all cases. To evaluate purity of the final product, the investigator should run different amounts of it on the same gel, from 5 μg, to visualize even minor impurities, to 0.2 μg to detect closely running proteolytic products.

Time Considerations There are several points in the expression and purification procedure where it can be paused and then resumed days or even weeks later. Cell growth and induction takes 1.5 days. It is important to resuspend the cells in the first portion of lysis buffer and incubate them with lysozyme immediately after obtaining the cell pellets. After that, the suspension must be frozen. It can then be stored up to at least 2 months at −80o C. This makes it feasible to express several proteins staggered by 1 day and accumulate numerous cell suspensions for future processing. The continuation of lysis, pelleting the debris, and protein precipitation by ammonium sulfate can be accomplished in a single day, with the resultant pellet stored for up to several weeks at −80°C. Subsequent steps, beginning with dissolving the pellet in MCB and all necessary chromatographic steps should be performed in close succession. Usually, it is most efficient to load the heparinSepharose column overnight, and then elute with a gradient, analyze the fractions, pool and concentrate the protein, and load the next column in a single day. For some proteins, it might be possible to load the next column in 2 to 3 hr, and then elute it with a gradient overnight. For others, loading overnight is necessary, with elution and fraction analysis occurring the next day. If the protein requires a third chromatographic step, an additional night of loading or gradient elution is needed, with fraction analysis and concentration carried out on the next day. Thus, purification of proteins that require two chromatographic steps takes 3 consecutive days. If loading of heparin-Sepharose begins on Monday, the next column is loaded on Tuesday, with Wednesday devoted to final fraction analysis, concentration, and dispensing of aliquots. Those purifications that require three steps take 1 extra day. The first day involves limited hands-on time, with the days devoted to analyzing fractions being more labor intensive.

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Acknowledgements This work was supported in part by NIH grants EY011500, GM077561, GM081756 (to V.V.G.), and GM095633 (to T.M.I.).

Literature Cited Ahmed, M.R., Zhan, X., Song, X., Kook, S., Gurevich, V.V., and Gurevich, E.V. 2011. Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination. Biochemistry 50:3749-3763. Attramadal, H., Arriza, J.L., Aoki, C., Dawson, T.M., Codina, J., Kwatra, M.M., Snyder, S.H., Caron, M.G., and Lefkowitz, R.J. 1992. βarrestin2, a novel member of the arrestin/βarrestin gene family. J. Biol. Chem. 267:1788217890. Chen, Q., Zhuo, Y., Kim, M., Hanson, S.M., Francis, D.J., Vishnivetskiy, S.A., Altenbach, C., Klug, C.S., Hubbell, W.L., and Gurevich, V.V. 2014. Self-association of arrestin family members. Handb. Exp. Pharmacol. 219:205-223. DeWire, S.M., Ahn, S., Lefkowitz, R.J., and Shenoy, S.K. 2007. β-arrestins and cell signaling. Annu. Rev. Physiol. 69:483-510. Donoso, L.A., Gregerson, D.S., Smith, L., Robertson, S., Knospe, V., Vrabec, T., and Kalsow, C.M. 1990. S-antigen: Preparation and characterization of site-specific monoclonal antibodies. Curr. Eye Res. 9:343-355. Gallagher, S. and Sasse, K. 1998. Protein analysis by SDS-PAGE and detection by Coomassie blue or silver staining. Curr. Protoc. Pharmacol. A.3B.1-A.3B.10 Gallagher, S.R., Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 2008. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 10.8.110.8.28. Goodman, O.B. Jr., Krupnick, J.G., Santini, F., Gurevich, V.V., Penn, R.B., Gagnon, A.W., Keen, J.H., and Benovic, J.L. 1996. β-arrestin acts as a clathrin adaptor in endocytosis of the β2-adrenergic receptor. Nature 383:447450. Gurevich, V.V. 1998. The selectivity of visual arrestin for light-activated phosphorhodopsin is controlled by multiple nonredundant mechanisms. J. Biol. Chem. 273:15501-15506. Gurevich, V.V. and Benovic, J.L. 2000. Arrestin: Mutagenesis, expression, purification, and functional characterization. Methods Enzymol. 315:422-437. Gurevich, E.V. and Gurevich, V.V. 2006a. Arrestins are ubiquitous regulators of cellular signaling pathways. Genome Biol. 7:236. Gurevich, V.V. and Gurevich, E.V. 2006b. The structural basis of arrestin-mediated regulation of G protein-coupled receptors. Pharm. Ther. 110:465-502. Arrestin Expression in E. coli and Purification

Gurevich, V.V. and Gurevich, E.V. 2014. Overview of different mechanisms of arrestin-mediated signaling. Curr. Protoc. Pharmacol. 67:2.10.12.10.9.

Gurevich, V.V., Chen, C.-Y., Kim, C.M., and Benovic, J.L. 1994. Visual arrestin binding to rhodopsin: Intramolecular interaction between the basic N-terminus and acidic C-terminus of arrestin may regulate binding selectivity. J. Biol. Chem. 269:8721-8727. Gurevich, V.V., Pals-Rylaarsdam, R., Benovic, J.L., Hosey, M.M., and Onorato, J.J. 1997. Agonistreceptor-arrestin, an alternative ternary complex with high agonist affinity. J. Biol. Chem. 272:28849-28852. Gurevich, V.V., Orsini, M.J., and Benovic, J.L. 1999. Characterization of arrestin expression and function. Recept. Biochem. Methodol. IV:157-178. Han, M., Gurevich, V.V., Vishnivetskiy, S.A., Sigler, P.B., and Schubert, C. 2001. Crystal structure of β-arrestin at 1.9 A: Possible mechanism of receptor binding and membrane translocation. Structure 9:869-880. Hanson, S.M., Francis, D.J., Vishnivetskiy, S.A., Klug, C.S., and Gurevich, V.V. 2006a. Visual arrestin binding to microtubules involves a distinct conformational change. J. Biol. Chem. 281:9765-9772. Hanson, S.M., Francis, D.J., Vishnivetskiy, S.A., Kolobova, E.A., Hubbell, W.L., Klug, C.S., and Gurevich, V.V. 2006b. Differential interaction of spin-labeled arrestin with inactive and active phosphorhodopsin. Proc. Natl. Acad. Sci. U.S.A. 103:4900-4905. Hanson, S.M., Cleghorn, W.M., Francis, D.J., Vishnivetskiy, S.A., Raman, D., Song, X., Nair, K.S., Slepak, V.Z., Klug, C.S., and Gurevich, V.V. 2007a. Arrestin mobilizes signaling proteins to the cytoskeleton and redirects their activity. J. Mol. Biol. 368:375-387. Hanson, S.M., Van Eps, N., Francis, D.J., Altenbach, C., Vishnivetskiy, S.A., Arshavsky, V.Y., Klug, C.S., Hubbell, W.L., and Gurevich, V.V. 2007b. Structure and function of the visual arrestin oligomer. EMBO J. 26:1726-1736. Hanson, S.M., Vishnivetskiy, S.A., Hubbell, W.L., and Gurevich, V.V. 2008. Opposing effects of inositol hexakisphosphate on rod arrestin and arrestin2 self-association. Biochemistry 47:10701075. Hirsch, J.A., Schubert, C., Gurevich, V.V., and Sigler, P.B. 1999. The 2.8 A crystal structure of visual arrestin: A model for arrestin’s regulation. Cell 97:257-269. Kim, M., Hanson, S.M., Vishnivetskiy, S.A., Song, X., Cleghorn, W.M., Hubbell, W.L., and Gurevich, V.V. 2011. Robust self-association is a common feature of mammalian visual arrestin1. Biochemistry 50:2235-2242. Kim, M., Vishnivetskiy, S.A., Van Eps, N., Alexander, N.S., Cleghorn, W.M., Zhan, X., Hanson, S.M., Morizumi, T., Ernst, O.P., Meiler, J., Gurevich, V.V., and Hubbell, W.L. 2012. Conformation of receptor-bound visual arrestin. Proc. Natl. Acad. Sci. U.S.A. 109:18407-18412. Kim, Y.J., Hofmann, K.P., Ernst, O.P., Scheerer, P., Choe, H.W., and Sommer, M.E. 2013.

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Crystal structure of preactivated arrestin p44. Nature 497:142-146. Kook, S., Zhan, X., Cleghorn, W.M., Benovic, J.L., Gurevich, V.V., and Gurevich, E.V. 2014. Caspase-cleaved arrestin-2 and BID cooperatively facilitate cytochrome C release and cell death. Cell Death Differ. 21:172-184. Laporte, S.A., Oakley, R.H., Zhang, J., Holt, J.A., Ferguson, S.S.G., Caron, M.G., and Barak, L.S. 1999. The 2-adrenergic receptor/arrestin complex recruits the clathrin adaptor AP-2 during endocytosis. Proc. Natl. Acad. Sci. U.S.A. 96:3712-3717. Lohse, M.J., Benovic, J.L., Codina, J., Caron, M.G., and Lefkowitz, R.J. 1990. β-arrestin: A protein that regulates β-adrenergic receptor function. Science 248:1547-1550. Luttrell, L.M., Ferguson, S.S., Daaka, Y., Miller, W.E., Maudsley, S., Della Rocca, G.J., Lin, F., Kawakatsu, H., Owada, K., Luttrell, D.K., Caron, M.G., and Lefkowitz, R.J. 1999. βarrestin-dependent formation of β2 adrenergic receptor-Src protein kinase complexes. Science 283:655-661. Lohse, M.J., Andexinger, S., Pitcher, J., Trukawinski, S., Codina, J., Faure, J.P., Caron, M.G., and Lefkowitz, R.J. 1992. Receptor-specific desensitization with purified proteins. Kinase dependence and receptor specificity of β-arrestin and arrestin in the β 2-adrenergic receptor and rhodopsin systems. J. Biol. Chem. 267:85588564. Olson, B.J.S.C. and Markwell, J. 2007. Assays for determination of protein concentration. Curr. Protoc. Pharmacol. 38:A.3A.1-A.3A.29. Palczewski, K. and Hargrave, P.A. 1991. Studies of ligand binding to arrestin. J. Biol. Chem. 266:4201-4206. Shukla, A.K., Manglik, A., Kruse, A.C., Xiao, K., Reis, R.I., Tseng, W.C., Staus, D.P., Hilger, D., Uysal, S., Huang, L.Y., Paduch, M., TripathiShukla, P., Koide, A., Koide, S., Weis, W.I., Kossiakoff, A.A., Kobilka, B.K., and Lefkowitz, R.J. 2013. Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature 497:137-141. S¨ohlemann, P., Hekman, M., Puzicha, M., Buchen, C., and Lohse, M.J. 1995. Binding of purified recombinant β-arrestin to guaninenucleotide-binding-protein-coupled receptors. Eur. J. Biochem. 232:464-472. Song, X., Vishnivetskiy, S.A., Gross, O.P., Emelianoff, K., Mendez, A., Chen, J., Gurevich, E.V., Burns, M.E., and Gurevich, V.V. 2009. Enhanced arrestin facilitates recovery and protects rod photoreceptors deficient in rhodopsin phosphorylation. Curr. Biol. 19:700-705. Song, X., Vishnivetskiy, S.A., Seo, J., Chen, J., Gurevich, E.V., and Gurevich, V.V. 2011. Arrestin-1 expression in rods: Balancing functional performance and photoreceptor health. Neuroscience 174:37-49. Song, X., Seo, J., Baameur, F., Vishnivetskiy, S.A., Chen, Q., Kook, S., Kim, M., Brooks,

E.K., Altenbach, C., Hong, Y., Hanson, S.M., Palazzo, M.C., Chen, J., Hubbell, W.L., Gurevich, E.V., and Gurevich, V.V. 2013. Rapid degeneration of rod photoreceptors expressing self-association-deficient arrestin-1 mutant. Cell Signal. 25:2613-2624. Sutton, R.B., Vishnivetskiy, S.A., Robert, J., Hanson, S.M., Raman, D., Knox, B.E., Kono, M., Navarro, J., and Gurevich, V.V. 2005. Crystal ˚ Evolution structure of cone arrestin at 2.3A: of Receptor Specificity. J. Mol. Biol. 354:10691080. Vishnivetskiy, S.A., Francis, D.J., Van Eps, N., Kim, M., Hanson, S.M., Klug, C.S., Hubbell, W.L., and Gurevich, V.V. 2010. The role of arrestin α-helix I in receptor binding. J. Mol. Biol. 395:42-54. Vishnivetskiy, S.A., Gimenez, L.E., Francis, D.J., Hanson, S.M., Hubbell, W.L., Klug, C.S., and Gurevich, V.V. 2011. Few residues within an extensive binding interface drive receptor interaction and determine the specificity of arrestin proteins. J. Biol. Chem. 286:24288-24299. Vishnivetskiy, S.A., Chen, Q., Palazzo, M.C., Brooks, E.K., Altenbach, C., Iverson, T.M., Hubbell, W.L., and Gurevich, V.V. 2013. Engineering visual arrestin-1 with special functional characteristics. J. Biol. Chem. 288:1174111750. Wu, N., Hanson, S.M., Francis, D.J., Vishnivetskiy, S.A., Thibonnier, M., Klug, C.S., Shoham, M., and Gurevich, V.V. 2006. Arrestin binding to calmodulin: A direct interaction between two ubiquitous signaling proteins. J. Mol. Biol. 364:955-963. Zhan, X., Gimenez, L.E., Gurevich, V.V., and Spiller, B.W. 2011a. Crystal structure of arrestin-3 reveals the basis of the difference in receptor binding between two non-visual arrestins. J. Mol. Biol. 406:467-478. Zhan, X., Kaoud, T.S., Dalby, K.N., and Gurevich, V.V. 2011b. Non-visual arrestins function as simple scaffolds assembling MKK4- JNK3α2 signaling complex. Biochemistry 50:1052010529. Zhan, X., Kaoud, T.S., Kook, S., Dalby, K.N., and Gurevich, V.V. 2013. JNK3 binding to arrestin-3 differentially affects the recruitment of upstream MAP kinase kinases. J. Biol. Chem. 288:2853528547. Zhan, X., Perez, A., Gimenez, L.E., Vishnivetskiy, S.A., and Gurevich, V.V. 2014. Arrestin-3 binds the MAP kinase JNK3α2 via multiple sites on both domains. Cell Signal. 26:766-776. Zhuang, T., Chen, Q., Cho, M.-K., Vishnivetskiy, S.A., Iverson, T.I., Gurevich, V.V., and Sanders, C.R. 2013. Involvement of distinct arrestin1 elements in binding to different functional forms of rhodopsin. Proc. Natl. Acad. Sci. U.S.A. 110:942-947. Zhuo, Y., Vishnivetskiy, S.A., Zhan, X., Gurevich, V.V., and Klug, C.S. 2014. Identification of receptor binding-induced conformational changes in non-visual arrestins. J. Biol. Chem. 289:20991-21002.

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