Functional analysis of human D1 and D5 dopaminergic G protein-coupled receptors: lessons from...

141

Nadine Kabbani (ed.), Dopamine: Methods and Protocols, Methods in Molecular Biology, vol. 964,DOI 10.1007/978-1-62703-251-3_10, © Springer Science+Business Media, LLC 2013

Chapter 10

Functional Analysis of Human D1 and D5 Dopaminergic G Protein-Coupled Receptors: Lessons from Mutagenesis of a Conserved Serine Residue in the Cytosolic End of Transmembrane Region 6

Bianca Plouffe and Mario Tiberi

Abstract

In mammals, dopamine G protein-coupled receptors (GPCR) are segregated into two categories: D1-like (D1R and D5R) and D2-like (D2R short , D2R long , D3R, and D4R) subtypes. D1R and D5R are primarily coupled to stimulatory heterotrimeric GTP-binding proteins (Gs/olf) leading to activation of adenylyl cyclase and production of intracellular cAMP. D1R and D5R share high level of amino acid identity in transmembrane (TM) regions. Yet these two GPCR subtypes display distinct ligand binding and G protein coupling properties. In fact, our studies suggest that functional properties reported for constitutively active mutants of GPCRs (e.g., increased basal activity, higher agonist af fi nity and intrinsic activity) are also observed in cells expressing wild type D5R when compared with wild type D1R. Herein, we describe an experimental method based on mutagenesis and transfection of human embryonic kidney 293 (HEK293) cells to explore the molecular mechanisms regulating ligand af fi nity, agonist-independent and dependent activity of D1R and D5R. We will demonstrate how to mutate one conserved residue in the cytosolic end of TM6 of D1R (Ser263) and D5R (Ser287) by modifying two or three nucleotides in the cDNA of human D1-like receptors. Genetically modi fi ed D1R and D5R cDNAs are prepared using a polymerase chain reac-tion method, propagated in E. coli , puri fi ed and mutations con fi rmed by DNA sequencing. Receptor expression constructs are transfected into HEK293 cells cultured in vitro at 37°C in 5% CO 2 environment and used in radioligand binding and whole cAMP assays. In this study, we will test the effect of S263A/G/D and S287A/G/D mutations on ligand binding and DA-dependent activation of D1R and D5R.

Key words: Dopamine , GPCR, D1-like receptors , Ligand binding , cAMP , Mutagenesis, Third intracellular loop , TM6 , HEK293 cells

Transmembrane (TM) signaling is fundamental in the homeostatic regulation of every major physiological function in eukaryotes ranging from yeast to human ( 1, 2 ) . A class of integral membrane

1. Introduction

142 B. Plouffe and M. Tiberi

proteins known as G protein-coupled receptors (GPCRs), which harbor seven a -helical TM segments, best illustrates this notion ( 3 ) . Indeed, over 800 types of GPCRs expressed throughout the body in humans are stimulated by a variety of ligands such as photons, protons, ions, odorants, lipids, biogenic amines, peptides and hor-mones ( 3 ) . Based on primary sequence and structural similarities, GPCRs can be grouped into fi ve families: rhodopsin (family A), secretin (family B), glutamate (family C), adhesion and Frizzled/Taste2 ( 3, 4 ) . Classically, heterotrimeric guanine-nucleotide-bind-ing proteins (G proteins) composed of a , b , and g subunits and located at the cytoplasmic side of plasma membrane, serve as molec-ular switches in GPCR signaling pathways by coupling ligand-induced receptor stimulation to intracellular responses. This is essentially accomplished by receptor activation of G proteins through the catalysis of GTP for GDP exchange on G a promoting a conformational change in GTP-bound G a and G b g subunits, which culminates in G protein subunit-mediated regulation of activity of different downstream effector proteins ( 3 ) . Meanwhile, studies also show that GPCR signaling can be mediated in a G protein-independent manner ( 5, 6 ) . Importantly, at least 50% of clinical drugs target GPCRs, hence highlighting their human thera-peutic relevance ( 7, 8 ) . Given the physiological and clinical impor-tance of these integral membrane proteins, it is crucial to know how the conserved seven- a -helix bundle structure imparts subtype-speci fi c ligand binding and activation properties to the GPCR members.

Difference in the extent of constitutive activity naturally dis-played by homologous GPCR subtypes was fi rst highlighted with D1-like dopaminergic receptors (D1R and D5R), which belong to the family A GPCRs ( 9 ) . In contrast to inhibitory G protein (Gi)-linked D2-like subtypes (D2R short , D2R long , D3R, D4R), D1R and D5R couple to stimulatory G proteins (Gs/olf) leading to adenylyl cyclase (AC) activation and production of intracellular cAMP ( 10, 11 ) . Interestingly, D5R naturally has a greater constitutive activity (i.e., increased ability to produce intracellular cAMP in the absence of agonists) when compared with D1R at similar receptor levels ( 9 ) . Furthermore, pharmacological properties of ligands displayed at D5R are highly reminiscent of those described for constitutively active mutant (CAM) GPCRs (e.g., higher agonist af fi nity and intrinsic activity) ( 9, 12 ) . The molecular and structural basis under-lying D1-like subtype-speci fi c functional properties has been dif fi cult to address experimentally as TM regions of D1R and D5R are almost indistinguishable (>80% identity) ( 13 ) . Indeed, the higher dopamine af fi nity of D5R relative to D1R cannot be explained by TM domains interacting with dopamine as critical amino acids implicated in catecholamine binding (e.g., TM5 serine residues) are conserved among dopaminergic and adrenergic recep-tors. Likewise, motifs found within cytosolic surfaces of TM

14310 Site-Directed Mutagenesis of D1 and D5 Dopaminergic Receptors

domains (e.g., “ionic lock” between TM3 and TM6) regulating receptor activation are also conserved among family A GPCRs ( 3 ) . This view is further underscored by data obtained from recent crystallographic studies of inactive states of different family A GPCRs, which display a highly conserved 7TM structural design ( 3 ) . Therefore, functional differences between GPCRs displaying high degree of TM identity likely implicate subtle variations in the assembly of conserved TM amino acids. These subtle variations are potentially mediated by GPCR subtype-speci fi c conformation of remote residues such as those found in receptor intracellular regions.

While amino acids located in receptor intracellular regions are unlikely to be directly involved in the binding to extracellular ligands, mutagenesis studies suggest that the third intracellular loop (IL3) and cytoplasmic tail (CT) play a major role in mediating D1-like subtype-speci fi c constitutive activity, agonist af fi nity and intrinsic activity ( 14– 18 ) . Notably, substitution of two variant resi-dues located in the cytosolic end of TM6 of D1R (F264 and R266) and D5R (I288 and K290) using site-directed mutagenesis show that F264 and I288 are major determinants in conferring D1-like subtype-speci fi c agonist binding, constitutive activity and agonist-dependent G protein-coupling properties ( 14 ) . Interestingly, F264 (D1R) and I288 (D5R) are located three amino acids downstream of the glutamate residue, which participates in the formation of an ionic lock with the highly conserved E/DRY motif of TM3. These D1R and D5R residues are not found in other catecholaminergic receptors and hence play potentially an important role in modulat-ing the arrangement of conserved amino acids forming the ionic lock. Herein, we describe an experimental approach to study the role of a conserved serine residue located adjacent to F264 (S263 in D1R) and I288 (S287 in D5R) (Fig. 1 ), which at this position does not exist in other catecholamine receptors. We test whether this conserved serine residue plays a critical role in the modulation of D1-like receptor binding and activation properties.

1. Custom DNA oligonucleotides (Sigma Genosys, Burlington, ON, Canada) as listed in Tables 1 and 2 . Lyophilized oligo-nucleotides are prepared as stock solutions in sterile Milli-Q water (resistivity of 18.2 M W cm) at 25 pmol/ m L for PCR primers or 2 pmol/ m L for DNA sequencing primer.

2. Expand High Fidelity Taq DNA polymerase (3.5 U/ m L), PCR buffer (10×), and MgCl 2 (25 mM) (Roche Diagnostics, Laval, QC, Canada). Store at −20°C.

2. Materials

2.1. Molecular Biology Reagents


3. PCR Nucleotides (dATP, dCTP, dGTP, and dTTP) at 100 mM (Fermentas, Burlington, ON, Canada). Store at −20°C.

4. dNTP Mix: Add 10 m L dATP, 10 m L dCTP, 10 m L dGTP, and 10 m L dTTP to 60 m L sterile Milli-Q water. Store at −20°C.

5. BoxI (PshAI), BsmI (Mva1269I), DraI, Eag I (Eco52I), EcoRI, HindIII, and XbaI restriction enzymes at 10 U/ m L with 10× Buffers (Y + /Tango TM or yellow; B + or blue; R + or red) from Fermentas. Store at −20°C.

6. Calf Intestinal Alkaline Phosphatase (CIAP) at 1 U/ m L and 10× dephosphorylation buffer from Fermentas. Store at −20°C.

7. T4 DNA ligase (5 U/ m L) and T4 DNA ligase buffer (10×) from Fermentas. Store at −20°C.

8. Tris–acetate–EDTA (TAE) buffer (50×): 2 M Tris–HCl, pH 8.0, 5.71% (v/v) glacial acetic acid, 0.5 M ethylenediamine tetraacetic acid (EDTA), pH 8.0 in Milli-Q water. Store at room temperature.

Fig. 1. Schematic representation of wild type hD1R and hD5R. Putative secondary structure of wild type hD1R and hD5R are indicated by circles . Distinct residues between hD1R and hD5R are depicted using black circles . Amino acid sequence of the region encompassing TM5, IL3, and TM6 is also shown. The mutated serine in IL3 of hD1R and hD5R is also indi-cated. hD1R human D1 receptor, hD5R human D5 receptor.


9. Agarose gels (1% w/v): Weigh out 0.5 g of agarose (Sigma-Aldrich, Oakville, ON, Canada) into a plastic 250 mL capped conical fl ask and add 50 mL of 1× TAE. Microwave for ~60 s with loosened cap. Gently swirl and microwave it again for 10 s and repeat 2–3 times if agarose is not completely dissolved. Keep watching agarose solution during microwaving as it can easily boil over. As the solution can become very hot, experi-menter should wear gloves and hold fl ask at arms length. Once agarose is completely dissolved let it cool down brie fl y (~5 min), add 2.5 m L of ethidium bromide (10 mg/mL) and gently swirl. Gloves should be worn when handling ethidium bro-mide solution as it is mutagenic and to some extent toxic. Slowly pour the agarose solution into a mini plastic casting tray, insert sample comb, push away any bubbles to the side using a pipet tip and allowed gel to solidify for 30–60 min at room temperature. Rinse out the fl ask (see Note 1 ).

10. 50% (v/v) Glycerol. Sterilize through 0.22 m m fi lter and store at room temperature.

Table 1 Sequences of oligonucleotide primers for the construction of single-point mutations of hD1R using a PCR-based overlapping approach

Construct Primer sequence 5 ¢ →3 ¢

hD1R-S263A P1 : TTcATcccAgTgcAgcTc P2 : TTcTcTTTT gAA T g C cATcTTAAAAgAAcTTTccgg P3 : TTTAAgATg G c A TTc AAAAgAgAAAcTAAAgTccTg P4 : TTAggAcAAggcTggTgg P5 : AcTgTgAcTccAgcc P6 : ggccAggAgAggcA

hD1R-S263D P1 : TTcATcccAgTgcAgcTc P2 : TTcTcTTTT A AA g TC cATcTTAAAAgAAcTTTccgg P3 : TTTAAgATg GA c TT T AAAAgAgAAAcTAAAgTccTg P4 : TTAggAcAAggcTggTgg P5 : AcTgTgAcTccAgcc P6 : ggccAggAgAggcA

hD1R-S263G P1 : TTcATcccAgTgcAgcTc P2 : TTcTcTTTT A AA g CC cATcTTAAAAgAAcTTTccgg P3 : TTTAAgATg GG c TT T AAAAgAgAAAcTAAAgTccTg P4 : TTAggAcAAggcTggTgg P5 : AcTgTgAcTccAgcc P6 : ggccAggAgAggcA

For each single-point mutant, a set of six primers numbered P1–P6 is used in PCR reactions. Nucleotide changes leading to S263A, S263D and S263G mutations are indicated in bold. Nucleotide changes intro-ducing diagnostic restriction sites through silent mutations are underlined or through the S263A mutation are underlined and bold


11. Loading Dye (10×): 0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanole FF (optional), 50% (v/v) glycerol in Milli-Q water. Store in aliquots at −20°C.

12. DNA Size Markers: Prepare 1× stock with 250 m L of One Kilobase Plus DNA Ladder (1 m g/ m L; Invitrogen, Burlington, ON, Canada), 250 m L of 10× loading dye, and 2,000 m L Milli-Q water. Store in aliquots at −20°C.

13. Sterile Luria Broth (LB) Medium: Weigh out 12.5 g of LB base (Invitrogen) into glass bottle or Fernbach fl ask (for DNA maxipreps) and add 500 mL of Milli-Q water. Sterilize liquid media using autoclave. Prior to autoclaving loosen cap on glass bottle and tape aluminum foil on top of fl ask. Always wear heat protective gloves when handling bottle or fl ask at the end of autoclave cycle. Store at room temperature. If store for a long time period, check that there is no microorganism contamina-tion prior to use.

Table 2 Sequences of oligonucleotide primers for the construction of single-point mutations of hD5R using a PCR-based overlapping approach

Construct Primer sequence 5 ¢ →3 ¢

hD5-S287A P1 : TAcggTgggAgg P2 : gATgg C AgcgcgcAg A cTggTgTcgggcgcgcAggc P3 : gcgcccgAcAccAg T cTgcgcgcT G ccATcAAgAAg P4 : TcATgTggATgTAggcAg P5 : AccTggccAAcTggA P6 : TgTTcAccgTcTccA

hD5-S287D P1 : TAcggTgggAgg P2 : gAT g TC AgcgcgcAg A cTggTgTcgggcgcgcAggc P3 : gcgcccgAcAccAg T cTgcgcgcT GA c ATcAAgAAg P4 : TcATgTggATgTAggcAg P5 : AccTggccAAcTggA P6 : TgTTcAccgTcTccA

hD5-S287G P1 : TAcggTgggAgg P2 : gAT g CC AgcgcgcAg A cTggTgTcgggcgcgcAggC P3 : gcgcccgAcAccAg T cTgcgcgcT GG c ATcAAgAAg P4 : TcATgTggATgTAggcAg P5 : AccTggccAAcTggA P6 : TgTTcAccgTcTccA

For each single-point mutant, a set of six primers numbered P1–P6 is used in PCR reactions. For each single-point mutant, a set of six primers numbered P1-P6 is used in PCR reactions. Nucleotide changes leading to S287A, S287D and S287G mutations are indicated in bold. Nucleotide changes introducing diagnostic restriction sites through silent mutations are underlined


14. Ampicillin (1,000×): 1 g of ampicillin (Sigma-Aldrich) is dissolved in Milli-Q water at 100 mg/mL and sterilized using a syringe fi lter (0.22 m m). Store in 0.5 mL aliquots at −20°C.

15. LB Ampicillin Plates: Weigh out 25 g of LB base (Invitrogen), 15 g of Bacto Agar (Fisher Scienti fi c, Ottawa, ON, Canada) into glass fl ask (for DNA maxipreps) and add 1 L of Milli-Q water. Sterilize using autoclave. Always wear heat protective gloves when handling fl ask at the end of autoclave cycle. Let it cool down for 15 min at room temperature. Thaw two 0.5 mL aliquots of ampicillin (1,000×), add to molten LB-agar, swirl, and pour into sterile polystyrene petri dishes (100 × 15 mm, Fisher Scienti fi c). Quickly pass over the petri dishes a fl ame from a Bunsen burner to remove bubbles. Keep moving fl ame while removing bubbles to avoid overheating LB-agar and break up ampicillin. 1 L of LB-agar will make 50 plates. Store inverted in plastic sleeves at 4°C for no longer than 3 months. Prior to using plates verify that there is no microorganism contamination.

16. SOB Medium: 2% (w/v) bacto-tryptone (Fisher Scienti fi c), 0.5% (w/v) bacto-yeast extract (Fisher Scienti fi c), and 10 mM NaCl. Sterilize by autoclaving. Store at room temperature. Prior to using SOB medium verify that there is no microorgan-ism contamination.

17. SOC Medium: SOB medium containing 10 mM MgCl 2 , 10 mM MgSO 4 , and 20 mM glucose. Solutions of 1 M MgCl 2 , 1 M MgSO 4 , and 2 M glucose are separately made and sterilize by fi ltration using 0.22 m m fi lter. Store at room temperature.

18. XL1-Blue Electroporation-Competent Cells (Agilent Technologies, Mississauga, ON, Canada). Store at −80°C.

19. Sterile Polypropylene Capped 13 mL Tubes (100 × 16 mm; Sarstedt, St-Léonard, QC, Canada).

20. Isobutanol (Fisher Scienti fi c). 21. Qiaex II Gel Extraction Kit (Buffer QX1, Buffer PE, Qiaex II

Bead Suspension) (Qiagen, Mississauga, ON, Canada). 22. QIAprep Spin Miniprep and Plasmid Maxiprep Kits from

Qiagen.

1. Adenovirus type 5-transformed human embryonic kidney 293 (HEK293) cells (CRL-1573, American Tissue Type Culture Collection, Manassas, VA).

2. Minimal Essential Medium (MEM) with Earle’s salt (Invitrogen).

3. Fetal Bovine Serum (FBS) (Invitrogen). Thaw frozen FBS bottle at 4°C overnight. The next day, warm up bottle in a 37°C water bath, heat-inactivate FBS in a 55°C water bath for

2.2. Cell Culture


1 h, and sterilize through 0.22 m m fi lter. Store in 25 and 50 mL aliquots in sterile capped polypropylene conical tubes at −20°C.

4. Gentamicin Sulfate (10 mg/mL) (Invitrogen). 5. Trypsin (0.25%) and EDTA (0.05% (w/v)) Buffer Solution

(Invitrogen). 6. Ca 2+ and Mg 2+ -free Phosphate-Buffered Saline (PBS) (Wisent,

St-Bruno, QC, Canada). 7. Tissue Culture Grade Sigma Hybri-Max Dimethylsulfoxide

(DMSO) (Sigma-Aldrich). Store at room temperature. 8. BD Falcon Polystyrene 75 cm 2 Flasks with 0.2 m m Vented Blue

Plug Seal Cap (VWR International, Montréal, QC, Canada). 9. Sterile HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfo-

nic acid) Buffer Solution (1 M, pH 7.4) (Invitrogen). 10. 20 mM HEPES-buffered MEM: In a certi fi ed biological safety

cabinet (BSC), add to bottle of MEM (500 mL), 10 mL of sterile HEPES (pH 7.4) and 0.5 mL of gentamicin (Sigma-Aldrich). Store at 4°C for up to 2 months.

11. 1 M HEPES (pH 7.0): 37.75 g of HEPES (Fisher Scienti fi c) is dissolved in 100 mL Milli-Q water, adjust pH to 7.0 and com-plete to 150 mL with Milli-Q water. Sterilize through 0.22 m m fi lter. Store in tissue culture area at room temperature (see Note 2 ).

12. 2 M NaCl: Prepare in glass bottle containing 500 mL of Milli-Q water and sterilize by autoclaving. Store in tissue culture area at room temperature.

13. 1 M Na 2 HPO 4 : Add 35.5 g of Na 2 HPO 4 in a beaker contain-ing 230 mL of Milli-Q water, stir on a hot plate at low temperature (setting 2) until completely dissolved, complete to a fi nal volume of 250 mL, and sterilize through 0.22 m m fi lter. Store in tissue culture area at room temperature.

14. 1 M NaH 2 PO 4 : Add 34.5 g of NaH 2 PO 4 in a beaker contain-ing 230 mL of Milli-Q water, stir on a hot plate at low tem-perature (setting 2) until completely dissolved, complete to a fi nal volume of 250 mL, and sterilize through 0.22 m m fi lter. Store in tissue culture area at room temperature.

15. 1 M Na 3 PO 4 : Prepare 25 mL by adding equal volumes of ster-ile 1 M Na 2 HPO 4 and 1 M NaH 2 PO 4 solutions in a BSC. Store in tissue culture area at room temperature (see Note 3 ).

16. Sterile 2× HEPES-buffered saline (0.28 M NaCl, 0.05 M HEPES, and 1.5 mM Na 3 PO 4 , pH 7.1): Add 70 mL of 2 M NaCl, 25 mL of 1 M HEPES, and 750 m L of 1 M Na 3 PO 4 to 400 mL of Milli-Q water in a glass beaker. Stir and adjust to pH 7.1 (±0.05). Complete to a fi nal volume of 500 mL and


sterilize through 0.22 m m fi lter in a BSC. Store in 15 mL ali-quots at −20°C.

17. Te fl on cell lifters (Fisher Scienti fi c).

1. [ 3 H]-SCH23390 and [ 3 H]-adenine (PerkinElmer NEN, Boston, MA). Store at −20°C (see Note 4 ).

2. Ascorbic acid, (+)-SCH23390 hydrochloride, dopamine hydrochloride, cis - fl upenthixol dihydrochloride and (+)-buta-clamol hydrochloride (Sigma-Aldrich). Store at room temper-ature (see Note 5 ).

3. Lysis Buffer: 10 mM Tris–HCl, pH 7.4 and 5 mM EDTA, pH 8.0. Store at 4°C.

4. Resuspension Buffer: 62.5 mM Tris–HCl, pH 7.4 and 1.25 mM EDTA, pH 8.0. Store at 4°C.

5. Binding Buffer: 62.5 mM Tris–HCl, pH 7.4 and 1.25 mM EDTA, pH 8.0, 200 mM NaCl, 6.7 mM MgCl 2 , 2.5 mM CaCl 2 and 8.33 mM KCl. Store at 4°C.

6. Washing Buffer (10×): 500 mM Tris–HCl, pH 7.4 and 1 M NaCl. Store at 4°C.

7. Whatman GF-C Glass fi ber Filter Sheets (Brandel Inc., Gaithersburg, MD).

8. Plastic Scintillation Vials (20 mL) (Sarstedt, Newton, NC). 9. Bio-Safe II Biodegradable Scintillation Cocktail (Research

Products International Corp., Mount Prospect, IL). 10. Bio-Rad Protein Assay Dye Concentrate (Bio-Rad Laboratories

Inc., Mississauga, ON, Canada). Store at 4°C. 11. Bovine Serum Albumin (BSA), minimum 96% electrophoresis

(Sigma-Aldrich). BSA is dissolved in sterile Milli-Q water at 1 mg/mL. Store in 1 mL aliquots at −20°C.

12. 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich) stored −20°C is dissolved in DMSO at 200 mM. Store stock solution at 4°C (see Note 6 ).

13. Neutralizing Solution: 4.2 M KOH. Store in a glass bottle at room temperature.

14. Cyclic AMP (cAMP) (Sigma-Aldrich). Store desiccated at −20°C.

15. [ 14 C]-cAMP (Moravek Biochemicals, Brea, CA). Store at −20°C.

16. cAMP Stop Solution: 2.5% (v/v) perchloric acid, 0.1 mM cAMP and [ 14 C]-cAMP (~3.3 nCi/mL; ~10,000 dpm) in 1,500 mL Milli-Q water. Store at 4°C (see Note 7 ).

17. Dowex AG 50 W-4X Resin (hydrogen form, 200–400 dry mesh, 63–150 m m wet beads) from Bio-Rad Laboratories (Hercules, CA).

2.3. Radioligand Binding and Whole Cell cAMP Assays


18. Alumina N Super I (MP Biomedicals, Montréal, QC, Canada). Store at room temperature.

19. Poly-Prep Columns (with 10 mL reservoir and graduated volume markings) from Bio-Rad Laboratories Inc.

20. Hydrochloric Acid (HCl) and Sodium Hydroxide (NaOH) at 0.1 N.

21. Imidazole (Sigma-Aldrich) is dissolved in Milli-Q water at 2 M, pH 7.5 (see Note 8 ). Store in plastic bottle at room tem-perature for up to 4 months.

The following experimental strategy describes how to generate three single-point mutants of S263 of hD1R (S263A, S263D and S263G) and S287 of hD5R (S287A, S287D, S287G) using a PCR-based overlap extension approach, DNA ligation, and DNA auto-mated sequencing (Fig. 2 ). The experimental approach entails two major steps:

1. DNA primers derived from forward and reverse nucleotide sequences coding for human D1R (hD1R) and D5R (hD5R) are designed to mutate the candidate serine into alanine, aspar-tate, and glycine. The hD1R and hD5R DNA templates are separately mixed with mutagenesis primers and a high proof-reading DNA polymerase to produce “megaprimers” using polymerase chain reaction (PCR) and an overlap extension approach. Pairs of puri fi ed overlapping “megaprimers” are subsequently fused together by PCR and puri fi ed by silica-gel particles to obtain the different mutated receptor DNA cas-settes. Mutated receptor DNA cassettes and corresponding wild type receptor DNA in the pCMV5 expression plasmid (cloning vector) are subjected to restriction digest and ligation procedures to generate different mammalian mutant receptor expression constructs.

2. Human embryonic kidney 293 (HEK293) cells are transfected with wild type and mutant receptor expression constructs using a DNA-calcium phosphate precipitation procedure, and seeded in tissue culture dishes and plates for radioligand binding and whole cell cAMP studies. Ligand binding properties of wild type and mutant receptors are measured in membrane preparations of transfected cells with saturation and competi-tion experiments using the D1-like selective radioligand [ 3 H]-SCH23390. Agonist-independent and dependent cou-pling to Gs is assessed in transfected cells metabolically labeled

3. Methods


with [ 3 H]-adenine and the amount of intracellular cAMP production determined from cell lysates with a sequential chro-matography puri fi cation procedure using Dowex and alumina columns. Radioligand binding and whole cell cAMP data are analyzed using nonlinear curve fi tting program and statistical tests. Our results suggest that S263 in hD1R and S287 in hD5R play a differential role in controlling ligand af fi nity and DA-induced activation of AC of human D1-like receptors (see data fi gures).

Fig. 2. Schematic representation of the making of pCMV5 expression constructs for single-point mutant forms of hD1R and hD5R. The key steps involved in the preparation of hD1R ( a ) and hD5R ( b ) single-point mutant constructs in the pCMV5 expression vector are shown (see text for details).


1. Qiagen maxipreps of wild type hD1R and hD5R subcloned in the expression vector pCMV5 are used as DNA templates (25 ng/ m L) in series of two PCR rounds as depicted in Fig. 3 . For each single-point mutant two PCR products (“megaprim-ers”), A and B, are separately ampli fi ed in the fi rst round using speci fi c set of P1–P2 and P3–P4 primer pairs (see Tables 1 and 2 ). First-round PCRs are carried out in a fi nal volume of 50 m L containing 50 ng of DNA template (2 m L of stock), 50 pmol of forward primer (2 m L of P1 or P3 stock solution at 25 pmol/ m L), 50 pmol of reverse primer (2 m L of P2 or P4 stock solution at 25 pmol/ m L), 1.5 mM MgCl 2 (3 m L of 25 mM stock solution), 0.2 mM dNTPs (1 m L of 10 mM stock solution), 3.5 U of Taq DNA polymerase (1 m L of stock

3.1. Preparation of Single-Point Mutant hD1R and hD5R Cassettes by Site-Directed Mutagenesis and PCR

Fig. 3. General scheme for creating single-point mutations using PCR-based overlapping approach. Representative exam-ple of the experimental strategy used to generate the S263G and S287G mutations in hD1R-pCMV5 ( a ) and hD5R-pCMV5 ( b ) constructs is depicted. This strategy remains identical for creating other single point mutations in hD1R (S263A and S263D) and hD5R (S287A and S287D). The beginning of the polylinker region of pCMV5 (EcoRI site) is arbitrarily set to position 0. Nucleotide position of 5 ¢ region of PCR primers (P1-P6) annealing to DNA expression construct template is indicated. Restriction sites used for generating mutated cassette are shown. The boundaries of mutated cassettes are illustrated using brackets.


solution), 5 m L of 10× PCR buffer and of 34 m L of sterile Milli-Q water. DNA is ampli fi ed in an Eppendorf Thermal Mastercycler using the following conditions: 1 cycle (94°C for 3 min, 50°C for 1 min, 72°C for 3 min), 24 cycles (94°C for 45 s, 50°C for 1 min, 72°C for 1 min) completed by an anneal extension step (50°C for 1 min and 72°C for 8 min). Importantly, during this fi rst PCR round, an overlapping region between A and B will be generated, which will allow amplifying the fi nal PCR product using primers P5–P6 during the second PCR round (see Subheading 3.1.5 ). At the end of the fi nal cycle, add 5.5 m L of 10× loading dye to PCR tubes.

2. Prepare 1× TAE buffer in Milli-Q water and pour a 1% (w/v) agarose minigel (see Subheading 2.1 , item 8). Once agarose solidi fi es, put casting tray in electrophoresis apparatus, fi ll with 1× TAE buffer, gently remove sample comb, and load wells with PCR samples (55 m L) and one well with 4 m L of DNA size markers. Separate samples for 1 h at 80 V and visualize ethidium bromide-stained DNA bands using an UV Transilluminator equipped with a digital camera.

3. Cut off appropriate sized DNA bands (see Fig. 3 ) and purify agarose-embedded DNA with QIAEX beads (Qiagen) accord-ing to manufacturer’s protocol. Elute puri fi ed bands from QIAEX beads using 20 m L of sterile Milli-Q water. Add 2 m L of puri fi ed bands to 7 m L of sterile Milli-Q water, mix with 1 m L of 10× loading dye and run samples beside a well loaded with 4 m L of DNA size markers on 1% (w/v) agarose minigel for 1 h at 80 V.

4. Visualize ethidium bromide-stained DNA bands using an UV Transilluminator equipped with a digital camera and take pic-ture to assist in the semi-quanti fi cation of puri fi ed products.

5. For the second-round PCRs, add equal volume of puri fi ed “megaprimer” A and B (~0.5 m L each) corresponding to the designated mutant receptor to a mix containing 19.5 m L of sterile Milli-Q water, 2.5 m L of 10× PCR buffer, 1.5 mM MgCl 2 (1.5 m L of 25 mM stock solution), 0.2 mM dNTPs (0.5 m L of 10 mM stock solution), and 1.75 U of Taq DNA polymerase (0.5 m L of stock solution). The overlap PCR is done in a fi nal volume of 25 m L using 1 cycle at 94°C for 3 min, 50°C for 1 min, 72°C for 10 min, and 20°C for 8 min.

6. During the 8-min period, 25 m L of a mix containing 19.5 m L of sterile Milli-Q water, 2.5 m L of 10× PCR buffer, 25 pmol of P5 forward primer (1 m L of 25 pmol/ m L stock solution), 25 pmol of P6 reverse primer (1 m L of 25 pmol/ m L stock solu-tion), 1.5 mM MgCl 2 (1.5 m L of 25 mM stock solution), 0.2 mM dNTPs (0.5 m L of 10 mM stock solution), and 1.75


U of Taq DNA polymerase (0.5 m L of stock solution) is added to overlap PCR mixture ( fi nal volume of 50 m L). PCR is then run for 25 cycles (94°C for 45 s, 50°C for 1 min, 72°C for 1 min) and completed by an anneal extension step (50°C for 1 min and 72°C for 8 min). At the end of this step, add 5.5 m L of 10× loading dye to PCR tubes.

7. Run fi nal PCR products on 1% (w/v) agarose and appropriate mutant receptor DNA cassettes puri fi ed as described (see Subheadings 3.1.2 , 3.1.3 , and 3.1.4 ).

1. Set up restriction enzyme digestions of the wild type hD1R-pCMV5 expression construct and puri fi ed mutated D1R DNA cassettes (849 bp) with HindIII and XbaI (see Fig. 3a ), and wild type hD5R-pCMV5 expression construct and puri fi ed mutated D5R DNA cassettes (569 bp) with BsmI and EagI (see Fig. 3b ) in a fi nal volume of 30 m L using separate auto-claved 1.5 mL Eppendorf tubes. Prepare reaction tubes for wild type hD1R and hD5R-pCMV5 expression constructs, in which restriction enzymes are replaced with an equivalent amount of sterile Milli-Q water. These tubes are referred to as uncut DNA (see Note 9 ).

2. For hD1R DNA digestions, add to tubes 8 m L of wild type hD1R-pCMV5 (0.125 m g/ m L working solution; 1 m g total) or puri fi ed mutant hD1R cassette (see Note 10 ), 16 m L of sterile Milli-Q water, 1.5 m L of HindIII (15 U), 1.5 m L of XbaI (15 U), and 3 m L of 10× Y + /Tango TM buffer (1× fi nal) (see Note 10).

3. For hD5R DNA digestions, add to tubes 8 m L of wild type hD5R-pCMV5 (0.125 m g/ m L working solution; 1 m g total) or puri fi ed mutant hD5R cassette, 13 m L of sterile Milli-Q water, 1.5 m L of BsmI (15 U), 1.5 m L of EagI (15 U), and 6 m L of 10× Y + /Tango TM buffer (2× fi nal) (see Note 11 ).

4. Gently pipette up and down to mix and fl oat tubes in a 37°C water bath for 1 h. At the end of incubation, put all tubes on ice (optional), add 3 m L of 10× loading dye to only the digested PCR products and mix by gently pipetting up and down.

5. Leave tubes containing digested hD1R-pCMV5 and hD5R-pCMV5 DNA constructs without loading dye and prepare linearized wild type hD1R and hD5R-pCMV5 DNA samples for dephosphorylation (see Note 12 ).

1. Carry out dephosphorylation reaction in using a fi nal volume of 50 m L. Add 14 m L of sterile Milli-Q water to tubes contain-ing the 30 m L of restriction digestion mix of hD1R-pCMV5 (HindIII-XbaI) and hD5R-pCMV5 (BsmI-EagI) expression constructs. Then, add 5 m L of 10× dephosphorylation buffer and 1 m L of CIAP (5 U).

3.2. Preparation of Linearized Wild Type Receptor Expression Constructs and Mutated Receptor DNA Cassettes by Digestions with Restriction Enzymes

3.3. Dephosphorylation of Linearized Wild Type hD1R and hD5R-pCMV5 DNA Constructs


2. Gently pipette up and down to mix and fl oat tubes in a 37°C water bath for 30 min.

3. At end of incubation, place dephosphorylation tubes on ice (optional), put in 5 m L of 10× loading dye and mix by gentle pipetting up and down.

1. Digested mutant cassettes and wild type expression constructs are loaded on individual wells along with a well containing 4 m L of DNA size markers of agarose minigels as described above in Subheading 3.1 (see Subheading 3.1.2 ). Run hD1R and hD5R samples for 1 h at 80 V on 1% (w/v) and 1.8% (w/v) agarose minigels, respectively (see Note 13). Visualize ethidium bromide-stained agarose gel using an UV Transilluminator equipped using a digital camera and excise appropriate bands: (1) HindIII-XbaI linearized hD1R-pCMV5 (~5,400 bp), (2) HindIII-XbaI digested mutant hD1RS263G cassette (745 bp), (3) HindIII-XbaI digested mutant hD1RS263A cassette (745 bp), (4) HindIII-XbaI digested mutant hD1RS263D cassette (745 bp), (5) BsmI-EagI linear-ized hD5R-pCMV5 (~6,000 bp), (6) BsmI-EagI digested mutant hD5RS287G cassette (348 bp), (7) BsmI-EagI digested mutant hD5RS287A cassette (348 bp), and (8) BsmI-EagI digested mutant hD5RS287D cassette (348 bp).

2. Purify agarose-embedded DNA bands with QIAEX beads (Qiagen) according to manufacturer’s protocol. Elute puri fi ed bands from QIAEX beads using 50 m L of sterile Milli-Q water or QIAEX elution buffer. Add 2 m L of puri fi ed bands to 7 m L of sterile Milli-Q water, mix with 1 m L of 10× loading dye and run samples beside a well loaded with 4 m L of DNA size mark-ers on 1% (w/v) agarose minigel prepared with thin sample comb for 1 h at 80 V.

3. Visualize ethidium bromide-stained DNA bands using an UV Transilluminator equipped with a digital camera and take pic-ture to assist in the semi-quanti fi cation of puri fi ed DNAs to set up ligations.

1. Thereafter and unless stated otherwise, linearized hD1R-pCMV5 (~5,400 bp band) and hD5R-pCMV5 (~6,000 bp band) will be called “vector” whereas the mutated receptor cassettes will be referred to as “inserts.” Set up control (vector alone) and test (vector + insert) ligation reactions in a fi nal vol-ume of 10 m L in 1.5 mL Eppendorf tubes on ice (optional). Add to test ligation tubes 0.5 m L vector, 0.5 m L insert (replace with 0.5 m L sterile Milli-Q water in control ligation tubes), 0.5 m L T4 DNA ligase (2.5 U), 1 m L 10× T4 DNA ligase buffer, and 7.5 m L sterile Milli-Q water (see Note 14 ).

3.4. Isolation of Linearized Wild Type hD1R and hD5R-pCMV5 DNA Constructs and Digested Mutant Receptor DNA Cassettes

3.5. DNA Ligation Reactions


2. Float tubes in a 16°C water bath overnight (see Note 15 ). Store ligation tubes at −20°C until use for transformation of XL-1 Blue electroporation-competent cells.

1. Add 40 m L of sterile Milli-Q water to fresh or thawed ligation tubes (10 m L) at room temperature and gently tap tubes to mix.

2. In a fume hood, add 500 m L of isobutanol to 50 m L ligation samples.

3. Mix by gently inverting tubes several times until isobutanol is fully miscible with aqueous ligation samples (no detection of isobutanol phase remnant or bubbles).

4. Spin tubes in a microfuge at 16,000 × g for 10 min at room temperature.

5. In a fume hood, decant supernatant in waste glass bottle and spin again tubes at 16,000 × g for 30 s.

6. In a fume hood, carefully remove supernatant using a P200 pipette and discard supernatant in waste glass bottle.

7. Let air dry the small DNA pellet in fume hood for 5–10 min, add 10 m L of sterile Milli-Q water to tubes and carefully resus-pend DNA pellet by washing sides of tubes.

1. Take 5 m L of desalted DNA samples from control and test ligation reactions and separately mix with 40 m L of XL1-Blue electroporation-competent cells on ice by gently pipetting up and down in 1.5 mL Eppendorf tubes.

2. Transfer 45 m L of DNA-bacteria mixtures into ice-cold elec-troporation cuvettes. Carefully wipe side of electroporation cuvettes to remove any condensation prior to inserting into electroporator.

3. Shock cells at 1,800 V for 5 ms (see Note 16 ). 4. Add 1 mL of freshly made SOC in each cuvette, gently pipette

up and down and transfer 1 mL to sterile polypropylene capped 13 mL tubes (100 × 16 mm).

5. Incubate with loosened cap in a 37°C shaking incubator at a velocity of 300 rpm for 1 h.

6. Pour bacterial cultures into 1.5 mL Eppendorf tubes and spin at 6,000 × g for 30 s at room temperature. Discard ~900 m L supernatant and gently resuspend bacterial pellet with leftover supernatant (~100 m L) by pipetting up and down.

7. Spread ~100 m L of bacterial cultures on pre-warmed (37°C) LB-ampicillin plates and grow bacteria in a 37°C incubator overnight (see Note 17 ).

3.6. Desalting of DNA Ligation Samples

3.7. Transformation of XL1-Blue Electroporation-Competent Cells with Ligated DNA Samples


1. Prepare sterile polypropylene capped 13 mL tubes containing 5 mL of LB with 1× ampicilin (100 m g/mL).

2. Pick two isolated colonies from each test ligation plates using small sterile pipette tips and inoculate a LB-ampicillin tube with a single colony by ejecting tip in it. Incubate tubes with loosened caps in a 37°C shaking incubator at a velocity of 300 rpm overnight.

3. Prepare backup bacterial glycerol stocks with overnight cultures. Add 0.7 mL of bacterial cultures to 0.3 mL of sterile 50% (v/v) glycerol ( fi nal concentration: 15% (v/v)) in 1.5 mL Eppendorf tubes and gently mix by inverting tubes several times. Snap-freeze in liquid nitrogen. Store at −80°C (see Note 18 ).

4. Make miniprep DNA with the rest of bacterial cultures (~4.3 mL) using QIAprep Spin Columns according to manu-facturer’s protocol (see Note 19 ).

5. Measure DNA concentration and purity of plasmid miniprep (total volume of 50 m L). Add 5 m L of plasmid miniprep DNAs in a fi nal volume of 1 mL of sterile Milli-Q water in quartz UV cuvettes. Read optical density (OD) against a blank solution (1 mL of sterile Milli-Q water) at wavelengths of 260 nm and 280 nm using spectrometer (see Note 20 ).

6. Set up diagnostic digestion reactions in a fi nal volume of 15 m L with and without appropriate restriction enzymes as follows (see Table 3 ).

7. For the screening of positive hD1R-S263A plasmid DNAs, carry out restriction digestions in 1.5 mL Eppendorf tubes containing 2 m L of plasmid DNA (0.5 m g; stock solution of 0.25 m g/ m L), 1.5 m L of 10× red buffer, 0.5 m L of BsmI (5U),

3.8. Preparation of Plasmid DNA Minipreps and Identi fi cation of Single-Point Mutants by Restriction Digestions

Table 3 Expected band size pattern of wild type and single-point mutants of hD1R and hD5R following digestion with restriction enzymes

Constructs Restriction enzymes Band sizes (base pairs)

hD1R-S263A BsmI + EcoRI Wild Type hD1R: 6025 Mutant hD1R: 804 , 5221

hD1R-S263G hD1R-S263D

DraI Wild Type hD1R: 19, 692, 1666, 3648 Mutant hD1R: 19, 692, 1112 , 1666, 2536

hD5R-S287A hD5R-S287G hD5R-S287D

BoxI Wild Type hD5R: 6322 Mutant hD5R: 612 , 5710

For each single-point mutant, the diagnostic band sizes of digested positive plasmid DNAs are bold and underlined


0.5 m L of EcoRI (5U), and 10.5 m L of sterile Milli-Q water (11.5 m L for uncut condition).

8. For the screening of positive hD1R-S263G and hD1R-S263D plasmid DNAs, carry out restriction digestions in 1.5 mL Eppendorf tubes containing 2 m L of plasmid DNA (0.5 m g; stock solution of 0.25 m g/ m L), 1.5 m L of 10× blue buffer, 0.5 m L of DraI (5U), and 11 m L of sterile Milli-Q water (11.5 m L for uncut condition).

9. For the screening of positive hD5R-S287A, hD5R-S287G and hD5R-S287D plasmid DNAs, carry out restriction digestions in 1.5 mL Eppendorf tubes containing 2 m L of plasmid DNA (0.5 m g; stock solution of 0.25 m g/ m L), 1.5 m L of 10× Y + /Tango TM buffer, 0.5 m L of BoxI (5U), and 11 m L of sterile Milli-Q water (11.5 m L for uncut condition).

10. Gently pipette up and down to mix and fl oat tubes in a 37°C water bath for 1 h. At the end of incubation, add 1.5 m L of 10× loading dye, mix by gently pipetting up and down and run samples beside a well loaded with 4 m L of DNA size markers on 1% (w/v) agarose minigel for 1 h at 80 V.

11. Visualize ethidium bromide-stained DNA bands using an UV Transilluminator equipped with a digital camera, take picture and identify positive mutant plasmid DNAs according to expected band sizes of digested wild type and mutated DNA (see Table 3 ).

1. Prepare samples for automated DNA sequencing as follows (see Note 21 ).

2. Make working solution of plasmid DNA minipreps at a fi nal concentration of 12.5 ng/mL with sterile Milli-Q water in autoclaved 1.5 mL Eppendorf tubes.

3. For each single-point mutants of hD1R, set up autoclaved 1.5 mL Eppendorf tubes containing 10 m L (125 ng) of plas-mid DNA and 5 m L (10 pmol) of hD1R-P1 forward primer or hD1R-P4 reverse primer (see Table 1 ) (see Note 22).

4. For each single-point mutants of hD5R, set up autoclaved 1.5 mL Eppendorf tubes containing 10 m L (125 ng) of plasmid DNA and 5 m L (10 pmol) of hD5R-P6 reverse primer (see Table 2 ).

5. Align sequenced DNAs with predicted nucleotide sequences and con fi rm that (1) serine is mutated, (2) mutated receptor DNA cassette is ligated in-frame with the expression vector pcCMV5 containing wild type hD1R or hD5R DNA sequences, and (3) integrity of restriction sites used for subcloning the mutated hD1R (HindIII and XbaI) and hD5R (BsmI and EagI) cassettes.

3.9. Preparation of Samples for Automated DNA Sequencing and Plasmid DNA Maxipreps


6. Once positive clone identify and DNA integrity con fi rmed, make large-scale plasmid DNA preparations as follows.

7. Stab glycerol stocks of positive bacterial clones with a sterile straight wire and streak pre-warmed (37°C) LB-ampicillin plates. Grow bacteria in a 37°C incubator overnight.

8. The next day, prepare sterile polypropylene capped 13 mL tubes containing 1 mL of LB with 1× ampicilin (100 m g/mL) and inoculate each tube with a single colony using sterile small pipette tips as described above (see Subheading 3.8 , step 2). Incubate tubes with loosened caps in a 37°C shaking incubator at a velocity of 300 rpm for 6 h.

9. Subsequently, add 10 m L of the small bacterial cultures to 1 mL of sterile LB in 1.5 mL Eppendorf tubes (see Note 23 ). Open aluminum foil on top of already made sterile Fernbach fl asks (see Subheading 2.1 , item 13) containing 500 mL of LB and 1× ampicillin with 1 mL of the diluted small bacterial cultures. Incubate fl asks in a 37°C shaking incubator at a velocity of 300 rpm overnight.

10. Prepare large-scale plasmid DNAs with Maxiprep Column Kit according to Qiagen’s protocol. Measure plasmid DNA concentration and purity (see Subheading 3.8 , step 5).

11. Verify integrity of plasmid receptor DNAs using digestions with restriction enzymes (see Subheading 3.8 , steps 6– 11 ).

1. Make frozen stocks of HEK293 cells in Nalgene cryovials at a density of 5 × 10 6 cells per mL of sterile freezing medium (10% tissue culture grade DMSO, 20% FBS, 70% MEM with Earle’s salts and 40 m g/mL gentamicin) (see Note 24 ).

2. Grow working stocks of HEK293 cells in polystyrene 75 cm 2 fl asks containing 20 mL of complete MEM (10% (v/v) FBS and 40 m g/mL gentamicin) at 37°C in a humidi fi ed 5% CO 2 incubator and maintain stocks as described previously ( 19 ) .

3. Prepare HEK293 cells for transfection as follows. 4. Aspirate medium from 75 cm 2 fl asks, add 5 mL of room

temperature PBS and wash cells by gently rocking fl asks (see Note 25).

5. Aspirate PBS, add 1 mL trypsin to fl asks, brie fl y incubate cells at room temperature (<1 min) (see Note 26 ).

6. Stop trypsinization by adding 20 mL of complete MEM per fl ask (see Note 27 ).

7. Mix cells by gentle trituration using 10 mL pipette. Pipette up and down 10–15 times to detach cells and avoid clump forma-tion. Count cells using hemacytometer.

3.10. Preparation and Transfection of HEK293 Cells


8. For the transfection procedure, seed polystyrene tissue culture dishes (100 × 20 mm) with 2 × 10 6 cells in fi nal volume of 10 mL of complete MEM and grow cells until the next day (24–30 h) at 37°C in a humidi fi ed 5% CO 2 incubator (see Note 28 ).

9. Prepare transfection solutions in sterile 13 mL polypropylene tubes (100 × 16 mm) in a BSC as follows.

10. Add a total quantity of 10 m g of different plasmid DNAs to be transfected in a fi nal volume of 50 m L or less of Milli-Q water to 13 mL tubes (see Note 29 ).

11. Add sterile Milli-Q water to each tube containing plasmid DNA to fi nal volume of 900 m L.

12. Add 100 m L of sterile 2.5 M CaCl 2 to each tube ( fi nal volume 1 mL) (see Note 30 ).

13. Add 1 mL of sterile 2× HEPES-buffered saline solution to DNA-calcium phosphate mixture in a dropwise manner using a P1000 pipette. Mix by gentle fl icking of tubes ( fi nal volume of 2 mL) (see Note 31 ).

14. Transfect two 100 × 20 mm dishes of HEK293 using 1 mL per dish as follows.

15. Open the lid of a dish, add dropwise 1 mL of transfection solution to the whole medium surface, close lid, and incubate HEK293 cells overnight at 37°C in humidi fi ed 5% CO2 incubator (see Note 32 ).

16. The next day, split cells as follows (see Note 33 ).

1. For each transfection condition, aspirate culture medium from the four dishes in a BSC, add 5 mL of room temperature PBS per dish to wash cells (see previous Note 25 ), aspirate PBS, add 0.5 mL of 1× trypsin per dish, incubate brie fl y, add 10 mL of complete MEM and triturate cells using gentle pipetting up and down (see Subheading 3.10 , step 7).

2. For radioligand binding studies, pool cells from four 100 × 20 mm dishes into a 150 × 25 mm dish ( fi nal volume ~40 mL) and grow transfected HEK293 cells for ~48 h at 37°C in humidi fi ed 5% CO2 incubator (see Note 34 ).

3. For dose–response curves using whole cell cAMP assays, pool cells from four 100 × 20 mm dishes ( fi nal volume ~40 mL) and seed two 12-well plates with 1 mL of cells per well (total vol-ume required is 24 mL) for each experimental condition. Seed also one 100 × 20 mm dish with 10–15 mL of cells per dish for determination of receptor levels in membrane preparations from cells used for dose–response curves (see Note 35 ).

3.11. Seeding of Transfected HEK293 Cells for Radioligand Binding and Whole Cell cAMP Assays


1. On the day of the experiment, put 150 × 25 mm dishes on ice, aspirate culture medium, add 10 mL of cold PBS to side of dishes and wash cells by gentle rocking of dishes.

2. Aspirate PBS, add 15 mL of ice-cold lysis buffer, detach cells with a cell lifter by scraping off the dish surface and transfer lysates to 50 mL polycarbonate centrifuge tubes (29 × 104 mm, Beckman Coulter). Wash dishes again with 15 mL of ice-cold lysis buffer, harvest 5 mL wash and put in centrifuge tubes.

3. Centrifuge samples at 40,000 g for 20 min at 4°C and put tubes on ice.

4. Discard supernatant, add 3 mL of ice-cold lysis buffer to centrifuge tubes, pipette up and down to detach pellets and homogenize pellets in centrifuge tubes with a Brinkmann Polytron at a velocity of 17,000 rpm for 15 s. Adjust fi nal volume in tubes to 30 mL and centrifuge at 40,000 g for 20 min at 4°C.

5. Discard supernatant, add 3 mL of ice-cold lysis buffer to cen-trifuge tubes, pipette up and down to detach pellets and homogenize pellets in centrifuge tubes with a Brinkmann Polytron at a velocity of 17,000 rpm for 15 s.

6. Add 0.6 mL of membrane preparations to tubes containing 3 mL of cold resuspension buffer (dilution factor of 1:6) and leave tubes on ice until used for saturation studies. Add the remnant of membrane preparations in lysis buffer in two 1.5 mL Eppendorf tubes (~1.2 mL per tube), snap-freeze in liquid nitrogen and store at −80 C until used for competition studies.

1. Set up four three-tier polypropylene racks (6 × 12 holes per row, Fisher Scienti fi c) containing 48 polystyrene test tubes (12 × 75 mm) to carry binding reactions in a fi nal volume of 500 m L (see Note 36 ).

2. Prepare six 10× concentrations of [ 3 H]-SCH23390 (84 Ci/mmol) in Milli-Q water ranging from ~0.1 to 100 nM ( fi nal in assays: ~0.01–10 nM) using a dilution factor of 1:3.

3. Prepare 10× cis - fl upenthixol (100 m M) in Milli-Q water using frozen stock ( fi nal in assays: 10 m M) (see Note 5 ).

4. Add 300 m L of binding buffer to all tubes ( fi nal in assays: 50 mM Tris–HCl, pH 7.4, 120 mM NaCl, 4 mM MgCl 2 , 1.5 mM CaCl 2 , 5 mM KCl, and 1 mM EDTA, pH 8.0).

5. Add 50 m L of Milli-Q water to total binding tubes or 50 m L of 10× cis - fl upenthixol (100 m M) to nonspeci fi c binding tubes.

6. Add 50 m L of different 10× [ 3 H]-SCH23390 concentrations to a set of four tubes from the lowest to highest concentration (see Note 36).

3.12. Preparation of Crude Membranes from Transfected HEK293 Cells for Saturation Studies

3.13. Saturation Studies


7. Add 100 m L of membrane preparations to tubes (24 tubes for each receptor tested), shake racks to mix and incubate at room temperature (~20°C) for 90–120 min.

8. Measure protein concentration in leftover membrane prepara-tions using Bio-Rad assay kit with BSA as standard according to manufacturer’s protocol (see Note 37 ).

9. Terminate binding reactions by rapid fi ltration through precut Whatmann GF/C glass fi ber fi lter sheets (11.4 cm × 31.1 cm) using Brandel semi-automated harvesting system (equipped with 48 harvesting probes) and wash membranes bound to fi lters three times with 5 mL of cold washing buffer (tubes are subjected to three fi ll up and aspiration cycles).

10. Put fi lter circles in plastic scintillation vials, add 5 mL of scintilla-tion cocktail and determine tritium-bound radioactivity in beta liquid scintillation counter with 30–40% counting ef fi ciency.

11. Analyze binding isotherms with a nonlinear curve- fi tting soft-ware from GraphPad Prism (GraphPad Software, San Diego, CA). Determine equilibrium dissociation constant ( K d , nM) and maximal binding capacity ( B max , pmol/mg membrane pro-teins) of [ 3 H]-SCH23390 with saturation curves (see Note 38 ). A representative example of saturation curves obtained in membrane preparations from transfected HEK293 cells with wild type (WT) and single-point mutant forms of hD1R and hD5R is shown in Fig. 4 . Averaged K d and B max values of [ 3 H]-SCH23390 are reported in Table 4 . Data are also expressed relative to hD1R-WT and hD5R-WT (Fig. 5 ). The S287A, S287G and S287D mutations have no signi fi cant effect on K d of [ 3 H]-SCH23390 while B max values are differentially modulated. Meanwhile, the S263G mutation in hD1R leads to a small but signi fi cant reduction of K d of [ 3 H]-SCH23390. This increased af fi nity of hD1R-S263G for [ 3 H]-SCH23390 is not observed in cells transfected with hD1R-S263A and hD1R-S263D. Moreover, while B max of hD1R-S263D is not changed relative to hD1R-WT, hD1R-S263G and hD1R-S263A dis-play a reduction and augmentation of B max, respectively.

1. Set up 16 three-tier polypropylene racks (6 × 12 holes per row, Fisher Scienti fi c) containing 48 polystyrene test tubes (12 × 75 mm) to carry binding reactions in a fi nal volume of 500 m L (see Note 39 ).

2. Thaw frozen tubes of membranes on ice and mix with 11 mL of resuspension buffer using Brinkmann Polytron at velocity of 17,000 rpm for 15 s. Leave membrane resuspension on ice until use (see Note 40 ).

3. Prepare a 10× concentration of [ 3 H]-SCH23390 (84 Ci/mmol) in Milli-Q water ranging from ~7 to 13 nM ( fi nal in assays: ~0.7–1.3 nM) ( see Note 41 ).

3.14. Competition Studies


Fig. 4. Saturation curves for HEK293 cells transfected with wild type and single-point mutant forms of hD1R and hD5R. Representative examples for saturation curves generating data for Table 4 are shown. Saturation curves of [ 3 H]-SCH23390 on crude membrane preparations were determined in the presence of 10 m M cis - fl upenthixol. TOTAL total binding, NS nonspeci fi c binding, hD1R human D1 receptor, hD5R human D5 receptor, WT wild type.


Tabl

e 4

Liga

nd b

indi

ng p

rope

rtie

s of

wild

type

and

sin

gle-

poin

t mut

ant f

orm

s of

dop

amin

e hD

1R a

nd h

D5R

Satu

ratio

n st

udie

s [ 3 H

]-SC

H233

90

Com

petit

ion

stud

ies

K i (nM

)

Rece

ptor

K d (

nM)

B max

(pm

ol/m

g pr

ot.)

SCH2

3390

Do

pam

ine

cis -

Flup

enth

ixol

(+

)-Bu

tacl

amol

hD1R

-WT

0.

82 (

0.64

–1.0

5)

13.5

(9.

2–17

.8)

0.80

(0.

56–1

.13)

7,

420

(4,0

19–1

3,69

9)

9.42

(5.

89–1

5.1)

5.

15 (

3.35

–7.9

3)

hD1R

-S26

3A

0.64

(0.

43–0

.96)

18

.7 (

12.5

–24.

9)

0.66

(0.

57–0

.78)

11

,085

(9,

273–

13,2

52)

8.37

(6.

04–1

1.6)

4.

98 (

3.51

–7.0

5)

hD1R

-S26

3G

0.59

(0.

40–0

.86)

7.

8 (5

.1–1

0.6)

0.

57 (

0.42

–0.7

7)

2,81

5 (2

,341

–3,3

85)

7.25

(5.

49–9

.58)

4.

39 (

3.46

–5.5

5)

hD1R

-S26

3D

0.69

(0.

55–0

.86)

13

.4 (

9.6–

17.2

) 0.

70 (

0.60

–0.8

2)

6,73

4 (5

,369

–8,4

46)

8.50

(6.

11–1

1.8)

4.

73 (

3.70

–6.0

3)

hD5R

-WT

1.

22 (

0.90

–1.6

5)

13.9

(10

.1–1

7.7)

0.

98 (

0.84

–1.1

5)

735

(542

–997

) 12

.1 (

8.69

–16.

9)

29.3

(18

.2–4

7.3)

hD5R

-S28

7A

1.03

(0.

87–1

.22)

17

.6 (

15.6

–19.

7)

0.91

(0.

75–1

.10)

91

6 (6

05–1

,388

) 11

.8 (

9.73

–14.

3)

24.7

(15

.4–3

9.4)

hD5R

-S28

7G

0.95

(0.

69–1

.31)

12

.1 (

10.3

–14.

0)

0.89

(0.

68–1

.15)

42

3 (3

14–5

72)

11.0

(9.

27–1

3.0)

23

.6 (

14.0

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4. Prepare 11 increasing 10× concentrations of SCH23390 (~3 × 10 −11 to 3 × 10 −6 M; ~3 × 10 −12 to 3 × 10 −7 M fi nal in assays), cis - fl upenthixol (~4 × 10 −10 to 5 × 10 −5 M; ~4 × 10 −11 to 5 × 10 −6 M fi nal in assays) and (+)-butaclamol (~3 × 10 −10 to 3 × 10 −5 M; ~3 × 10 −11 to 3 × 10 −6 M fi nal in assays) in Milli-Q water using frozen stock ( see Note 5 ).

5. Prepare a fresh 10× ascorbic acid solution (1 mM) in Milli-Q water.

6. Prepare 11 increasing 10× concentrations of dopamine (~3 × 10 −8 to 1 × 10 −2 M; fi nal in assays) in 10× ascorbic acid solution ( see Note 42 ).

7. Add 300 m L of binding buffer ( fi nal in assays: 50 mM Tris–HCl, pH 7.4, 120 mM NaCl, 4 mM MgCl 2 , 1.5 mM CaCl 2 , 5 mM KCl, and 1 mM EDTA, pH 8.0).

8. Add 50 m L of Milli-Q water or 10× ascorbic acid to total binding tubes.

Fig. 5. Relative equilibrium dissociation constant ( K d ) and maximal binding capacity ( B max ) values of hD1R and hD5R single-point mutants. Arithmetic means ± S.E. of K d ( a ) and B max ( b ) values of hD1R and hD5R single-point mutants were calculated rela-tive to their respective wild type receptor counterpart. * P < 0.05 when compared with a value of 1 (wild type receptor) using one-sample t test. hD1R-WT wild type human D1 receptor, hD5R-WT wild type human D5 receptor.


9. Add 50 m L of different 10× concentrations of cold drugs from the lowest to highest concentration.

10. Add 50 m L of the fi xed 10× concentration of [ 3 H]-SCH23390.

11. Add 100 m L of membrane preparations to tubes (96 tubes for each receptor tested), shake racks to mix and incubate at room temperature (~20°C) for 90–120 min.

12. Terminate binding reactions by rapid fi ltration through precut Whatmann GF/C glass fi ber fi lter sheets (11.4 cm × 31.1 cm) using Brandel semi-automated harvesting system and wash membranes bound to fi lters three times with ~5 mL of cold washing buffer (tubes are subjected to three fi ll up and aspira-tion cycles).

13. Put fi lter circles in plastic scintillation vials, add 5 mL of scintil-lation cocktail and determine tritium-bound radioactivity in beta liquid scintillation counter with 30–40% counting ef fi ciency.

14. Analyze binding isotherms using GraphPad Prism software. Determine equilibrium dissociation constant of cold (unla-beled) drugs ( K i , nM). A representative example of competi-tion curves obtained in membrane preparations from transfected HEK293 cells with WT and single-point mutant forms of hD1R and hD5R is depicted in Fig. 6 . Averaged K i values of unlabeled drugs are reported in Table 4 . Data are also expressed relative to hD1R-WT and hD5R-WT (Fig. 7 ). None of the mutations has a major effect on the af fi nity of cis - fl upenthixol and (+)-butaclamol, two inverse agonists. Interestingly, in both hD1R and hD5R, the S-to-G mutation leads to a signi fi cant increase in dopamine af fi nity (lower K i value) of receptors while S-to-D mutation had no effect (Table 4 ). Additionally, we observe that the S-to-A mutation promotes a decrease in dopamine af fi nity of hD1R and hD5R, a trend that is not how-ever statistically signi fi cant. Importantly, K i values measured with unlabelled SCH23390 are in line with K d values of [ 3 H]-SCH23390 (Table 4 ). It is also worth mentioning that the increased af fi nity of hD1R-S263G for [ 3 H]-SCH23390 was also observed with unlabelled SCH23390 using competi-tion studies (Table 4 ).

1. Prepare MEM containing 5% (v/v) FBS and 40 m g/mL gen-tamicin in a BSC and add sterile [ 3 H]-adenine (stock at 1 mCi/mL) using a dilution factor 1:1,000 to obtain a fi nal activity of 1 m Ci/mL.

2. Aspirate medium from 12-well plates, add 1 mL of prewarmed (37°C) labeling media per well and incubate HEK293 cells with [ 3 H]-adenine overnight at 37°C in humidi fi ed 5% CO2 incubator ( see Note 43 ).

3.15. Establishment of Dopamine Dose–Response Curves using Whole Cell cAMP Assays


Fig. 6. Competition curves for HEK293 cells transfected with wild type and single-point mutant forms of hD1R and hD5R. Representative examples for competition curves generating data for Table 4 are shown. Competition curves on crude membrane preparations from wild type and single-point mutant forms of hD1R ( left panels ) and hD5R ( right panels ) were performed with 0.5–1.2 nM of [ 3 H]-SCH23390 in the absence and presence of increasing concentrations of unlabeled drugs. Concentration of unlabeled drugs ( M ) is given as log values. hD1R-WT wild type human D1 receptor, hD5R-WT wild type human D5 receptor.

Fig. 7. Relative equilibrium dissociation constant of unlabeled drug ( K i ) values of hD1R and hD5R single-point mutants. Arithmetic means ± S.E. of K i values of hD1R and hD5R single-point mutants were calculated relative to their respective wild type receptor counterpart. * P < 0.05 when compared with a value of 1 (wild type receptor) using one-sample t test. hD1R-WT wild type human D1 receptor, hD5R-WT wild type human D5 receptor.


3. Prepare two scintillation vials containing 50 m L of labeling media and 5 mL of scintillation cocktail. Count in a beta liquid scintillation counter ( see Note 44 ).

4. After an overnight labeling with [ 3 H]-adenine, perform whole cell cAMP assays as follows.

5. Thaw 200 mM IBMX stock and prepare 20 mM HEPES-buffered MEM containing 1 mM IBMX. Keep in a 37°C water bath until use.

6. Prepare racks of polystyrene test tubes (12 × 75 mm) containing 100 m L of neutralizing solution.

7. Prepare a fresh 100× ascorbic acid solution (10 mM) in Milli-Q water.

8. Prepare seven increasing 100× concentrations of dopamine (10 −9 to 10 −3 M; 10 −11 to 10 −5 M fi nal in assays) in 100× ascorbic acid solution using 1:10 dilution factor (see Note 42).

9. Aspirate labeling medium and add 1 mL of 20 mM HEPES-buffered MEM containing 1 mM IBMX per well.

10. Add 10 m L of 100× ascorbic acid to three wells (triplicate determinations).

11. Add 10 m L of increasing 100× concentrations of dopamine in triplicate to remaining wells of the two 12-well plates.

12. Incubate 12-well plates at 37°C for 30 min ( see Note 45 ). 13. At the end of treatment period, put 12-well plates on ice tray,

aspirate medium, add 1 mL of cAMP stop solution and incubate cells at 4°C for 30 min.

14. Transfer lysates from wells to tubes containing neutralizing solution, mix using vortex and put a para fi lm sheet to seal top of tubes in racks. Store at 4°C until use ( see Note 46 ).

15. Prepare membranes from the binding dish as follows. 16. Put dishes on ice tray, aspirate culture medium and add 5 mL

of cold PBS to side of dishes and wash by gentle rocking of dishes.

17. Aspirate PBS, add 5 mL lysis buffer, detach cells with a cell lifter by scraping off the dish surface and transfer lysates to a 15 mL polycarbonate centrifuge tubes (18 × 100 mm, Beckman Coulter). Wash dishes again with 5 mL of lysis buffer and trans-fer whole volume to centrifuge tubes ( fi nal volume of 10 mL). Prepare membranes essentially as described in Subheading 3.12 ( steps 3–6 ). Resuspend fi nal pellets in 0.6–1 mL of resuspen-sion buffer and perform binding reactions using 100 m L of membranes and a saturating concentration of [ 3 H]-SCH23390 in the absence and presence of cold 10 m M cis - fl upenthixol as described above ( see Subheading 3.13 ). Measure protein concentrations with the Bio-Rad assay kit with BSA as standard to determine B max in pmol/mg membrane proteins.


1. Mount Bio-Rad Poly-Prep columns on custom-made Plexiglas racks (100 per rack), fi ll separate set of columns with Dowex and alumina and establish elution pro fi le of columns using 0.1 N NaOH/ 0.1 N HCl/distilled water (Dowex) and 0.1 M imidazole (alumina) as previously described ( 19 ) .

2. Prepare KOH-neutralized cAMP samples for sequential chro-matography as follows.

3. Prepare also triplicate samples of 1 mL of cAMP stop solution in tubes containing neutralizing solution, mix using vortex and store with [ 3 H]-cAMP lysate samples at 4°C ( see Note 47 ).

4. Centrifuge test tubes containing KOH-neutralized cAMP samples at ~500 g (1,500 rpm) in Beckman Coulter Allegra TM 6R Centrifuge for 10 min ( see Note 48 ).

5. Load 0.85 mL of samples on separate Dowex columns and let drain the fl ow through fraction ( see Note 49 ).

6. Add 1 mL of distilled water to each column, let drain the water wash fraction from Dowex and elute samples from Dowex on alumina columns using 4 mL of distilled water.

7. Let drain water wash fraction from alumina, wash with 1 mL of 0.1 M imidazole and let drain the imidazole wash fraction.

8. Elute samples from alumina columns into plastic scintillation vials using 3 mL of 0.1 M imidazole. Add 18 mL of scintilla-tion cocktail. Shake vials vigorously to mix aqueous samples (~3 mL) with scintillation cocktail and count vials in beta liq-uid scintillation counter using dual 3 H and 14 C channels ( see Note 50 ).

9. Take a 50 m L aliquot from leftover volume (~200 m L) of each KOH-neutralized cAMP lysate samples, add to plastic scintilla-tion vials, fi ll vials with 5 mL of scintillation cocktail and count in beta liquid scintillation counter using dual 3 H and 14 C channels ( see Note 51 ).

10. Enter 3 H and 14 C counts in Excel spreadsheet and compute intracellular [ 3 H]-cAMP levels in the absence and presence of increasing concentrations of dopamine using formulas described in Plouffe et al. ( 19 ) .

11. Compute intracellular cAMP levels expressed in arbitrary units (CA/TU × 1,000) and analyze averaged dose–response curves with a four-logistic parameter equation using a nonlinear curve- fi tting program from GraphPad Prism to determine effective concentration of dopamine that produces 50% of maximal stim-ulation (EC 50 , an index of potency) and dopamine-mediated maximal stimulation of AC ( E max ). The best- fi tted dose–response curves to dopamine obtained with cells expressing similar recep-tor levels are reported in Fig. 8 . Our data show that S-to-A, S-to-G, and S-to-D mutations modulate EC 50 and E max .

3.16. Puri fi cation of [ 3 H]-cAMP with Sequential Chromatography on Dowex and Alumina Columns and Quanti fi cation of Intracellular [ 3 H]-cAMP Mediated by Dopamine


Fig. 8. DA-induced formation of intracellular cAMP by wild type and single-point mutant forms of hD1R and hD5R in intact HEK293 cells. Cells were grown in medium containing 1 m Ci/mL of [ 3 H]-adenine overnight. Intracellular cAMP levels were determined in 12-well dishes in the absence and presence of increasing concentration of DA (10 −11 to 10 −5 M). Each point represents the arithmetic mean ± S.E. of three to four experiments done in triplicate determinations. ( a ) Dose–response curves to DA were simultaneously analyzed by a four-parameter logistic equation using GraphPad Prism version 5.03 using constrained or unconstrained parameters. Best- fi tted values for effective concentration that elicits 50% of maximal stimu-lation (EC 50 , nM) with 95% lower and upper con fi dence intervals are as follows: 14.7 (6.87–31.4) for hD1R, 43.8 (18.5–104) for hD1R-S263A, 9.90 (5.20–18.9) for hD1R-S263G, 30.1 (13.2–68.4) for hD1R-S263D, 1.50 (0.64–3.49) for hD5R, 3.15 (1.11–8.94) for hD5R-S287A, 0.72 (0.28–1.85) for hD5R-S287D, and 1.96 (0.68–4.98) for hD5R-S287G. ( b ) Best- fi tted values for E max ± S.E. obtained from dose–response curves to dopamine in HEK293 cells expressing wild type and single-point mutant forms of hD1R and hD5R are shown. ( c ) EC 50 shift (expressed as arithmetic mean ± S.E.) of single-point mutants were calculated relative to wild type receptor using EC 50 values derived from individual fi tted dose–response curves. * P < 0.05 when compared with wild type receptor. The B max values in pmol/mg/membrane proteins (expressed as arithmetic mean ± S.E.) are as follows: 1.04 ± 0.18 (hD1R-WT), 1.34 ± 0.32 (hD1R-S263A), 1.19 ± 0.35 (hD1R-S263G), 1.15 ± 0.20 (hD1R-S263D), 1.12 ± 0.34 (hD5R), 1.31 ± 0.28 (hD5R-S287A), 0.83 ± 0.04 (hD5R-S287G), and 1.04 ± 0.26 (hD5R-S287D). hD1R-WT wild type human D1 receptor, hD5R-WT wild type human D5 receptor.


Interestingly, the trend in mutation-speci fi c effects is generally recapitulated in cells expressing hD1R and hD5R mutants (Fig. 8 ). Data obtained with the S-to-G mutation are in agree-ment with the increased dopamine af fi nity for hD1R-S263G and hD5R-S287G (Table 4 and Fig. 7 ). The S-to-G mutation may thus promote the release of molecular constraints within the cytosolic end of TM6, leading to a more “relaxed” confor-mation for agonist binding and agonist-dependent G protein coupling. This idea is to some extent supported by our binding data showing increased af fi nity of hD1R-S263G for SCH23390 (Figs. 6 and 7 ). Indeed, SCH23390 is a classical D1-like antag-onist that behaves pharmacologically as a partial agonist in HEK293 cells ( 9, 20 ) . In addition, the decreased potency (EC 50 rightward shift) observed with the S-to-A mutation (Fig. 8 ) suggests a potential role of the hydroxyl group on the side chain of serine in the formation of hydrogen bonds, which may be only involved in agonist-dependent G protein coupling. Indeed, in contrast to S-to-G mutation, the S-to-A mutation has no effect on agonist binding (Table 4 and Fig. 7 ). Therefore, S-to-A mutation may impart subtle structural changes to the cytosolic end of TM6 that speci fi cally impact the agonist-dependent G protein-coupling conformation. Meanwhile, the S-to-D mutation in hD1R (and to a lesser extent in hD5R) paradoxically increases EC 50 (lower potency, i.e., reduced G protein coupling) and E max (greater dopamine ef fi cacy) (Fig. 8 ). These results may imply that the phosphomimetic S-to-D mutation promotes the formation of an ionic bond between the negatively charged side chain in aspartic acid and a posi-tively charged amino acid (arginine, histidine or lysine). Ultimately, this may lead to the formation of a new GPCR conformation with distinct G protein coupling properties when compared with wild type receptors. However, it remains to be established whether results obtained with the phosphomimetic S-to-D mutation can also be recapitulated with the phosphory-lation of S263 and S287 in hD1R and hD5R, respectively. In closing, it is also worth mentioning that the role of S263 (hD1R) and S287 (hD5R) in regulating the constitutive activity will require additional studies. In fact, dose–response curves in 12-well dishes do not provide the best experimental frame-work to address this important question ( see Note 52 ).

1. Ethidium bromide wastes (tip, gel) should be disposed as per the experimenter’s research institution rules.

2. The 1 M HEPES solution is utilized to prepare 2× HEPES-buffered saline solution, which is used in the transfection

4. Notes


procedure. Note that the pH of this HEPES solution is different from that of the sterile HEPES buffer solution (pH 7.4) employed in the preparation of 20 mM HEPES-buffered MEM (whole cAMP assays).

3. Na 3 PO 4 precipitates can form over time. Precipitates can be easily dissolved by swirling solution in a 37°C water bath prior to preparing stock of 2× HEPES-buffered saline solution.

4. Handling and disposal of radioactive material should be done as per radiation safety of fi ce rules of the experimenter’s research institution.

5. Experimenter can make stock solution of cold drugs in Milli-Q water (SCH23390, cis - fl upenthixol) or ethanol ((+)-butacla-mol) at a fi nal concentration of 10 mM. Store stock solution of drugs at −20°C. However, ascorbic acid and dopamine solu-tions are always made fresh.

6. The IBMX-DMSO stock solution will freeze at 4°C. The 200 mM IBMX is made in a 50 mL polypropylene conical tube, which facilitates rapid thawing in 37°C water bath. We recommend preparing small volume of solution, typically using 5 mL of DMSO, to avoid multiple “freeze–thaw” cycles. The IBMX stock solution should be discarded if experimenters note that it is unfrozen when stored at 4°C.

7. Typically, stop solution is prepared in large polypropylene graduated cylinder (2 L) and carefully poured in two dark glass bottles (~750 mL) fi tted with a bottletop dispenser. The stop solution can be kept for 4 months at 4°C. Unlabelled cAMP is used to saturate phosphodiesterases and hence to prevent deg-radation of [ 3 H]-cAMP formed by receptor stimulation. [ 14 C]-cAMP is used as a tracer to measure [ 3 H]-cAMP recov-ery during chromatography column procedure.

8. The imidazole working solution is diluted in Milli-Q water at 0.1 M. 0.1 M imidazole is stored at room temperature in a glass bottle equipped with bottletop dispenser and used during alumina column washing and elution procedure.

9. The electrophoresis of uncut DNA is recommended and useful in assessing the ef fi ciency of digestion with selected restriction enzymes and isolation of positive clones for single-point mutants.

10. The volume of puri fi ed mutant receptor cassette can be increased to 16 m L if the recovery yield of mutant receptor cas-sette is low after puri fi cation using QIAEX beads.

11. Double digestions using restriction enzymes from Fermentas are typically performed in either 2× or 1× buffer Y + /Tango TM (yellow) according to optimal enzyme activity. For a double digestion using BsmI and EagI, 2× buffer Y + /Tango TM gives the highest enzyme activities.


12. Do not add loading dye following digestion of hD1R-pCMV5 and hD5R-pCMV5 DNA constructs because it will compromise CIAP dephosphorylation. This step is critical for the ligation reaction. It allows dephosphorylation of 5 ¢ -ends of digested constructs DNA that have not be fully cut with both enzymes and hence reducing the self-ligation of single enzyme-digested DNA molecules during ligation reactions. Ultimately, this will decrease the number of false positive on your test ligation plates.

13. A fraction of the hD5R DNAs is partially digested with BsmI and EagI. It is thus recommended to run side by side uncut and cut samples on 1.8% (w/v) agarose gel to visualize and isolate DNA bands fully digested by BsmI and EagI.

14. We usually prepare ligation reactions using a vector and insert ratio of 1:3. The volume of vector and insert used in our liga-tion reactions is based on relative size and intensity between vector and insert bands. If yield of puri fi ed vector is far in excess of your puri fi ed insert, we recommend adding 0.5 m L of a 1:10 dilution of the puri fi ed vector in sterile Milli-Q water.

15. Typically, a water bath is placed in cold room or chromatogra-phy refrigerator (4°C) and temperature of water bath is set to reach 16°C.

16. Optimal desalting is critical for electroporation of bacteria. If high salt concentrations remain in your ligation reactions, this will create an electrical discharge or arcing during electropora-tion. Arcing will signi fi cantly decrease the viability of bacteria and ultimately lead to a poor ef fi ciency of transformations.

17. We generally obtain less than 5 colonies following transforma-tion with control ligations while test ligation yield between 25 and 50 colonies. The rate of positive colonies ranged between 80 and 90%.

18. The preparation of bacterial glycerol stocks of selected colonies prior to making plasmid DNA minipreps for analysis using restriction digestions and automated DNA sequencing will avoid retransforming bacteria with the positive plasmid DNA following the con fi rmation of the positive mutation. Overall, having readily available bacterial glycerol stocks will save the experimenter from redoing steps described in Subheading 3.7 and thus work more ef fi ciently.

19. Alternatively, boiling methods can be used to prepare plasmid DNA minipreps. The boiling methods are perhaps more cost-ef fi cient in comparison to using QIAprep Spin columns and as suitable for restriction digestions. However, in our experience, boiling methods do not give high enough DNA quality for automated DNA sequencing and thus the experimenter will have to rely on other methods to make higher quality plasmid


DNA preps. In our opinion, the cost does not justify using boiling methods.

20. Concentrations of plasmid DNA minipreps range from 0.25 to 0.5 m g/ m L with a purity (OD 260 /OD 280 ratio) of ~1.8–1.9.

21. Automated DNA sequencing protocol may vary according to company or core facility’s protocol. Make sure that method described herein is suitable with core facility or company per-forming automated DNA sequencing.

22. If the same oligonucleotides is used as PCR and sequencing primers, experimenter will make sure that primers are made at appropriate concentrations for PCR and sequencing reactions.

23. This step does not require the addition of ampicillin as diluted bacterial cultures are immediately inoculated into LB contain-ing 1× ampicillin.

24. ATTC suggest that HEK293 cells be grown in 15% (v/v) horse serum. However, we have replace horse serum with heat-inac-tivated FBS without aberrant effect on cell growth.

25. Do not add PBS directly on cells as they may detach from fl asks before adding trypsin.

26. You can gently rock fl asks to accelerate trypsinization of cells. 27. Prior to begin cell culture work, put trypsin and complete

MEM 37°C water bath to warm up solutions. When ready to start cell culture, trypsin and MEM bottles are wiped with 70% (v/v) ethanol and put in level 2-certi fi ed biological safety cabi-net (BSC). Trypsin and MEM can be left at room temperature in BSC during cell culture procedures.

28. Dishes can also be seeded at a cell density up to 2.5 × 10 6 cells/dish. If you observed that cells seeded at 2.5 × 10 6 cells/dish grow too fast at high number of cell passages (>P48), we rec-ommend that dishes be seeded at a lower cell density (2 × 10 6 cells). In our experience, cells transfected at a density ranging from 2 to 2.5 × 10 6 cells does not signi fi cantly impact the D1-like receptor expression. However, our unpublished data suggest that when cells are seeded at higher cell density than 2.5 × 10 6 cells/dish (>3 × 10 6 cells/dish), there is a greater vari-ability in receptor expression. Experimenter should bear in mind that the aforementioned guidelines are those that have been optimal for our laboratory.

29. For radioligand binding studies, we typically transfect each dish of cells with 5 m g of plasmid DNA. In our hands, this amount of DNA yields in transfected HEK293 cells the maximal achievable receptor expression as measured with [ 3 H]-SCH23390 (~15–20 pmol/mg membrane proteins).


Indeed, the use of higher amounts of DNA (10–20 m g/dish) does not lead to greater expression levels of wild type (WT) or low-expressing mutant forms of hD1R or hD5R. With respect to performing dose–response curves in intact cells, we titrate the amount of receptor expression construct DNA to be used in transfection to achieve lower levels of receptor (1–3 pmol/mg membrane proteins). For instance, the amounts of receptor expression construct DNA used herein for dose–response curves are as follows: hD1R-WT (0.04–0.07 m g/dish), hD1R-S263A (0.04 m g/dish), hD1R-S263G (0.08–12 m g/dish), hD1R-S263D (0.06–0.08 m g/dish), hD5R-WT (0.04–0.08 m g/dish), hD5R-S287A (0.06 m g/dish), hD5R-S287G (0.06–0.08 m g/dish), and hD5R-S287D (0.08 m g/dish). Importantly, when using lower amount of 5 m g/dish of receptor expression construct DNAs, the total amount of plasmid DNA must be normalized at a constant amount per transfected dish (5 m g) using empty plasmid (e.g., pCMV5). This will mitigate variations in transfection ef fi ciency between different condi-tions. The ef fi ciency in HEK293 cells obtained using our trans-fection method will not be covered here as this issue has been previously discussed elsewhere ( 17, 19 ) .

30. Remove drips left on side of tubes by gentle tapping on a hard surface inside the BSC.

31. The pH of 2× HEPES-buffered saline solution (pH to 7.1 ± 0.05) is critical for optimal transfection of plasmid DNA. Higher and lower pH will signi fi cantly impact the formation DNA-calcium phosphate precipitates. For instance, we observed a drastic reduction in receptor expression with a solution at pH of 7.3.

32. We observe that the proliferation rate of HEK293 cells slowly get higher up to 52 passages under our cell culture conditions. After 52 passages, HEK293 cells grow more rapidly and some-times as foci. In addition, cells adhere less on dishes at older passages. In our laboratory, we generally utilize HEK293 cells between 40 and 52 passages.

33. Typically, we use four transfection dishes per experimental condition in radioligand binding and whole cell cAMP assays. Meanwhile, the number of transfected dishes can be scaled up if mutant receptors display low expression levels in transfected HEK293 cells (<1 pmol/mg membrane proteins) or if larger amounts of membrane preparations are needed for radioligand binding assays.

34. For radioligand binding studies, we normally let transfected HEK293 cells grow for 48 h following seeding in 150 × 25 mm dishes. For instance, if the transfection is performed on Tuesday, cells are split and seeded in new dishes on Wednesday and then


used to make membrane preparations on Friday. Alternatively, the experimenter can use cells on Thursday (cells grown for 24 h) as we observe that expression is high enough to detect receptor binding.

35. Cells in 100 × 20 mm dishes are used to prepare crude mem-branes to determine receptor levels for each transfection condi-tion using radioligand binding.

36. In general, 4 rows of 12 holes are used to set up two saturation curves with six concentrations of [ 3 H]-SCH23390 using dupli-cate determinations for total and nonspeci fi c binding (24 tubes for each receptor tested) for a total of 48 tubes per rack. This set up is optimal for terminating reactions using our Brandel harvesting system. For the purpose of our study, four racks were used to perform saturation studies with wild type and single-point mutants of hD1R and hD5R. A set of four tubes is used for each concentration of [ 3 H]-SCH23390 (two tubes for total binding followed by two tubes for nonspeci fi c binding).

37. We usually carry out assays with 25–100 m L of membrane resuspension in a fi nal volume of 800 m L of Milli-Q water in glass test tubes (10 × 75 mm) and 200 m L of protein dye. Mix well by vortexing several times.

38. Bmax value is used as an index of total receptor levels. 39. Typically, one rack (4 rows of 12 holes) allows performing two

competition curves. For the purpose of the present study, each receptor construct to be tested requires two racks of 48 tubes to test four dopaminergic compounds (SCH23390, dopamine, cis - fl upenthixol, (+)-butaclamol). Each competition curve is done in the absence (total binding) and presence of 11 increas-ing concentrations of cold drugs using duplicate determina-tions (24 tubes for each drug tested).

40. Volume of resuspension can be reduced to increase the speci fi c binding signal if receptor constructs are expressed at a B max lower than 1 pmol/mg of membrane proteins. If so, the num-ber of drugs to be tested will be reduced accordingly. Alternatively, to test four drugs in the same experiment, one can prepare a larger number of frozen stocks of membrane preparations of the given receptor expressed at low B max .

41. Competition curves are performed using a fi xed concentration of [ 3 H]-SCH23390 similar to K d values measured at wild type and mutant receptors.

42. Competition studies and dose–response curves with dopamine are done in the presence of ascorbic acid at a fi nal concentration of 0.1 mM in the assays to prevent dopamine oxidation.


43. Values obtained from dose–response curves remain essentially unchanged whether HEK923 cells are metabolically labeled with 1 or 2 m Ci/mL of [ 3 H]-adenine. Therefore, the use of 1 m Ci/mL of [ 3 H]-adenine will considerably decrease the amount of handled radioactivity and cost of experiments. Make also a note that if cells are not labeled on the following day of seeding, cells can be labeled the same day of experiment using an amount of ³ 2 m Ci/mL of [ 3 H]-adenine to increase signal detection. Under these circumstances, cells are metabolically labeled for a minimum of 4 h prior to doing whole cell cAMP assays.

44. These samples are used to assess the amount of [ 3 H]-adenine in labeling media. Vials can be put aside until counting the other vials with radiolabeled eluates obtained from double column chromatography.

45. Cells can also be incubated with drugs for a shorter time period.

46. Samples can be stored at 4°C up to 5 days. In our hands, no differences have been measured between samples processed immediately for double chromatography columns and samples processed after being left at 4°C for 5 days. Samples should be frozen at −20°C if longer time of storage is required. However, experimenter should fi rst verify that the used test tubes do not crack −20°C.

47. The average [ 14 C]-cAMP counts from triplicate samples (column input) will be computed to determine the column recovery and establish a correction factor (CF) for each set of paired Dowex and alumina columns. The column input (C14 total ) is calculated from counting the radioactivity in vials containing 0.85 mL of KOH-neutralized cAMP stop solution mixed with 2 mL of 0.1 M imidazole (to make a 3 mL aqueous sample) and 18 mL of scintillation cocktail.

48. Samples are clari fi ed using a low-speed centrifugation to pellet down potassium perchlorate salts prior to applying samples on Dowex columns. Loading salts on Dowex columns will inter-fere with sample elution and lead potentially to spurious radio-active counts.

49. We choose to apply a volume of 0.85 mL on Dowex columns to prevent touching and pipetting salt precipitates from the bottom of test tubes.

50. While the volumes for washes and elution of samples on Dowex and alumina columns reported herein are those routinely obtained in our laboratory, it is strongly recommended that the column pro fi les be thoroughly reassessed in the experimenter’s laboratory as described ( 19 ) .


51. [ 3 H] counts measured in 50 m L aliquots will be used to determine the total amount of [ 3 H]-adenine uptake (TU) in each well (an index of cell number).

52. The assessment of constitutive activity of wild type and mutant forms of hD1R and hD5R is beyond the scope of this study. The determination of constitutive activity of wild type and mutant forms of hD1R and hD5R is typically performed with cells seeded in 6-well plates and incubated in labeling medium containing 2 m Ci/mL of [ 3 H]-adenine overnight at 37°C in humidi fi ed 5% CO2 incubator. Generally, cells used for these assays express receptors at higher B max values. Methods pertain-ing to the study of constitutive activity of D1-like receptors and their mutants have been discussed elsewhere ( 19 ) .

Acknowledgments

We would to thank Dr. Kursad Turksen for his advice and Andrew Charrette for reading the manuscript. Bianca Plouffe was a recipi-ent of a graduate scholarship from Fonds de la recherche en santé du Québec. This work was supported by an operating grant from Canadian Institutes of Health Research (MOP-81341).

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