Dopamine Receptor Oligomerization Visualized in Living Cells
2005; Elsevier BV; Volume: 280; Issue: 44 Linguagem: Inglês
10.1074/jbc.m504562200
ISSN1083-351X
AutoresBrian F. O’Dowd, Ji X, Mohammad Alijaniaram, Ryan D. Rajaram, Michael M. C. Kong, Asim J. Rashid, Tuan Nguyen, Susan R. George,
Tópico(s)Neuropeptides and Animal Physiology
ResumoG protein-coupled receptors occur as dimers within arrays of oligomers. We visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor to another cellular compartment. We incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. (+)-Butaclamol retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. Agonist-nonbinding D1(S198A/S199A)-NLS, containing two substituted serine residues in transmembrane 5 also oligomerized with D1, and both were retained on the cell surface by (+)-butaclamol. Drug removal disrupted these oligomerized receptors so that D1 remained at the cell surface while D1(S198A/S199A)-NLS trafficked to the nucleus. Thus, receptor conformational differences permitted oligomer disruption and showed that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. We demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. However, D2 did not heterooligomerize with the structurally modified D1(S198A/S199A), indicating an impaired interface for their interaction. Thus, we describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it. G protein-coupled receptors occur as dimers within arrays of oligomers. We visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor to another cellular compartment. We incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. (+)-Butaclamol retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. Agonist-nonbinding D1(S198A/S199A)-NLS, containing two substituted serine residues in transmembrane 5 also oligomerized with D1, and both were retained on the cell surface by (+)-butaclamol. Drug removal disrupted these oligomerized receptors so that D1 remained at the cell surface while D1(S198A/S199A)-NLS trafficked to the nucleus. Thus, receptor conformational differences permitted oligomer disruption and showed that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. We demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. However, D2 did not heterooligomerize with the structurally modified D1(S198A/S199A), indicating an impaired interface for their interaction. Thus, we describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it. G protein-coupled receptors (GPCRs) 3The abbreviations used are:GPCRG protein-coupled receptorNLSnuclear localization signalBTCbutaclamol(+)BTC and (-)BTC(+)- and (-)-butaclamol respectivelyGFPgreen fluorescent proteinRFPred fluorescent proteinmRFPmonomerized red fluorescent proteinIC1-2 and -3intracellular loop 1 2 and 3HAhemagglutininFITCfluorescein isothiocyanate. 3The abbreviations used are:GPCRG protein-coupled receptorNLSnuclear localization signalBTCbutaclamol(+)BTC and (-)BTC(+)- and (-)-butaclamol respectivelyGFPgreen fluorescent proteinRFPred fluorescent proteinmRFPmonomerized red fluorescent proteinIC1-2 and -3intracellular loop 1 2 and 3HAhemagglutininFITCfluorescein isothiocyanate. form dimers and higher order oligomers, as inferred from a large body of evidence garnered from a variety of methodological approaches (1George S.R. O'Dowd B.F. Lee S.P. Nat. Rev. Drug Discovery. 2002; 1: 808-820Crossref PubMed Scopus (533) Google Scholar, 2Milligan G. Mol. Pharmacol. 2004; 66: 1-7Crossref PubMed Scopus (420) Google Scholar, 3Bouvier M. Nat. Rev. Neurosci. 2001; 2: 274-286Crossref PubMed Scopus (578) Google Scholar), and their static configuration has been visualized by atomic force microscopy in the case of rhodopsin to reveal dimers arranged in clustered rows, with each row accommodating 10-20 dimers (4Fotiadis D. Liang Y. Filipek S. Saperstein D.A. Engel A. Palczewski K. Nature. 2003; 421: 127-128Crossref PubMed Scopus (657) Google Scholar, 5Liang Y. Fotiadis D. Filipek S. Saperstein D.A. Palczewski K. Engel A. J. Biol. Chem. 2003; 278: 21655-21662Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar). Rhodopsin-related GPCRs function as arrays of oligomers (5Liang Y. Fotiadis D. Filipek S. Saperstein D.A. Palczewski K. Engel A. J. Biol. Chem. 2003; 278: 21655-21662Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 6Lee S.P. O'Dowd B.F. Ng G.Y. Varghese G. Akil H. Mansour A. Nguyen T. George S.R. Mol. 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The structural details involved in the formation of receptor dimers or oligomers have not been elucidated, with little experimental evidence for fundamental questions, such as their behavior at the cell surface, whether the oligomers remain intact or separate, and if homooligomers and heterooligomers behave differently. That the oligomerized GPCR structures modulate the properties and conformations of the individual constituent receptors involved has been shown for μ- and δ-opioid receptor heterooligomers (8George S.R. Fan T. Xie Z. Tse R. Tam V. Varghese G. O'Dowd B.F. J. Biol. Chem. 2000; 275: 26128-26135Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 11Gomes I. Jordan B.A. Gupta A. Trapaidze N. Nagy V. Devi L.A. J. Neurosci. 2000; 20 (20, Rapid Communication110, 1-5): 1-5Crossref PubMed Google Scholar, 12Levac B.A. O'Dowd B.F. George S.R. Curr. Opin. Pharmacol. 2002; 2: 76-81Crossref PubMed Scopus (122) Google Scholar) and D1 and D2 dopamine receptor heterooligomers (13Lee S.P. So C.H. Rashid A.J. Varghese G. Cheng R. Lanca A.J. O'Dowd B.F. George S.R. J. Biol. Chem. 2004; 279: 35671-35678Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). In these structures, the ligand binding properties and/or the coupling properties were altered, depending on whether a receptor was within a homooligomer or heterooligomer. G protein-coupled receptor nuclear localization signal butaclamol (+)- and (-)-butaclamol respectively green fluorescent protein red fluorescent protein monomerized red fluorescent protein intracellular loop 1 2 and 3 hemagglutinin fluorescein isothiocyanate. G protein-coupled receptor nuclear localization signal butaclamol (+)- and (-)-butaclamol respectively green fluorescent protein red fluorescent protein monomerized red fluorescent protein intracellular loop 1 2 and 3 hemagglutinin fluorescein isothiocyanate. The biophysical techniques utilized to investigate GPCR oligomers, such as bioluminescence resonance energy transfer (14Gales C. Rebois R.V. Hogue M. Trieu P. Breit A. Hebert T.E. Bouvier M. Nat. Methods. 2005; 2: 177-184Crossref PubMed Scopus (324) Google Scholar) or fluorescence resonance energy transfer (9McVey M. Ramsay D. Kellett E. Rees S. Wilson S. Pope A.J. Milligan G. J. Biol. Chem. 2001; 276: 14092-14099Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 15Rocheville M. Lange D.C. Kumar U. Patel S.C. Patel R.C. Patel Y.C. Science. 2000; 288: 154-157Crossref PubMed Scopus (740) Google Scholar), permitted the analysis of receptor-receptor or receptor-protein interactions in situ, within living cells. However, these techniques still have only limited ability to investigate the many aspects of oligomer structure or function that still remain unknown. There is a need to provide insight into the questions regarding receptor oligomeric complexes, such as (i) the precise sites of interactions maintaining monomers in a dimer formation; (ii) whether true heterodimers exist or only homodimers within a heterooligomeric complex; (iii) whether the homodimer interactions differ from the heterodimer; (iv) the numbers of dimers in an oligomer; and (v) whether oligomers form larger complexes and can be functionally regulated. Thus, elucidation of the mechanism underlying the formation of oligomeric structures, analysis of their functional properties, or analysis of their behavior in cells requires new experimental paradigms. We wished to explore some of the above aspects of oligomerization to understand the dynamics governing the formation and trafficking of oligomers, to ultimately understand the functional relevance of oligomers in cellular processes. We devised a strategy that engineered the trafficking of a GPCR to another cellular compartment and hypothesized that if it took with it its oligomeric partner, this would provide definitive proof of oligomerization and provide a tool to study its dynamics in the cell. We and others determined that homodimerization in the rhodopsin-like GPCRs utilizes a transmembrane domain dimer interface (6Lee S.P. O'Dowd B.F. Ng G.Y. Varghese G. Akil H. Mansour A. Nguyen T. George S.R. Mol. Pharmacol. 2000; 58: 120-128Crossref PubMed Scopus (140) Google Scholar, 16Lee S.P. O'Dowd B.F. Rajaram R.D. Nguyen T. George S.R. Biochemistry. 2003; 42: 11023-11031Crossref PubMed Scopus (120) Google Scholar, 17Guo W. Shi L. Javitch J.A. J. Biol. 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Only a few GPCRs contain endogenous NLSs (20Lu D. Yang H. Shaw G. Raizada M.K. Endocrinology. 1998; 139: 365-375Crossref PubMed Google Scholar, 21Chen R. Mukhin Y.V. Garnovskaya M.N. Thielen T.E. Iijima Y. Huang C. Raymond J.R. Ullian M.E. Paul R.V. Am. J. Physiol. 2000; 279: F440-F448Crossref PubMed Google Scholar, 22Watson P.H. Fraher L.J. Natale B.V. Kisiel M. Hendy G.N. Hodsman A.B. Bone. 2000; 26: 221-225Crossref PubMed Scopus (57) Google Scholar). One example, the angiotensin AT1 receptor, contains an endogenous NLS in helix 8, which serves to direct the receptor into the nucleus in certain cells (10Lee D.K. Lanca A.J. Cheng R. Nguyen T. Ji X.D. Gobeil Jr., F. Chemtob S. George S.R. O'Dowd B.F. J. Biol. Chem. 2004; 279: 7901-7908Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 20Lu D. Yang H. Shaw G. Raizada M.K. Endocrinology. 1998; 139: 365-375Crossref PubMed Google Scholar). The NLS strategy was evaluated using dopamine receptors. Dopamine D1 and D2 receptors each have been shown to form homooligomers and together to form heterooligomers (13Lee S.P. So C.H. Rashid A.J. Varghese G. Cheng R. Lanca A.J. O'Dowd B.F. George S.R. J. Biol. Chem. 2004; 279: 35671-35678Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 23Ng G.Y.K. Trogadis J. Stevens J. Bouvier M. O'Dowd B.F. George S.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10157-10161Crossref PubMed Scopus (72) Google Scholar, 24Ng G.Y. O'Dowd B.F. Lee S.P. Chung H.T. Brann M.R. Seeman P. George S.R. Biochem. Biophys. Res. Commun. 1996; 227: 200-204Crossref PubMed Scopus (243) Google Scholar). We were the first to demonstrate that D2 receptors exist as homodimers in human and rat brain (25Zawarynski P. Tallerico T. Seeman P. Lee S.P O'Dowd B.F. George S.R. FEBS Lett. 1998; 441: 383-386Crossref PubMed Scopus (117) Google Scholar), and our demonstration of D1 and D2 receptor complexes by co-immunoprecipitation from rat brain and heterologous cells demonstrated that these receptors heterooligomerize (13Lee S.P. So C.H. Rashid A.J. Varghese G. Cheng R. Lanca A.J. O'Dowd B.F. George S.R. J. Biol. Chem. 2004; 279: 35671-35678Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). The functional synergism between D1 and D2 dopamine receptors was evidenced by the generation of a novel calcium signal by receptor coactivation (13Lee S.P. So C.H. Rashid A.J. Varghese G. Cheng R. Lanca A.J. O'Dowd B.F. George S.R. J. Biol. Chem. 2004; 279: 35671-35678Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). By fluorescence resonance energy transfer analysis, we revealed that the D1 and D2 receptors exist in close proximity on the cell surface, presumably within a heterooligomeric complex. Furthermore, the D1 and D2 receptor heterooligomers displayed novel agonist-induced internalization and trafficking patterns, distinct from that of D1 and D2 receptor homooligomers (26So C.H. Varghese G. Curley K.J. Kong M.M. Alijaniaram M. Ji X. Nguyen T. O'Dowd B.F. George S.R. Mol. Pharmacol. 2005; 68: 568-578Crossref PubMed Scopus (91) Google Scholar). In this report, we show that incorporating an NLS into several of the dopamine receptors mediated receptor translocation to the nucleus. We used this translocation strategy as outlined (Fig. 1a) to understand the ability of these GPCRs to co-traffic with their oligomerization partners as a test of the robustness of the interaction between them and to probe the structural conformation of homo- and heterooligomers after ligand occupancy and after introduction of structural variation by point mutagenesis. The method enabled the identification of both homo- and heterooligomers for the dopamine receptors and demonstrated that both types of interactions were robust enough to result in co-trafficking of oligomeric partners to the nucleus. Conformational homogeneity of the receptors was necessary to maintain the integrity of a homooligomer, and the interaction between similar receptors within a homomeric structure could not be disrupted. Furthermore, the introduction of any structural dissimilarity of the receptors within the homooligomer, whether induced by drug occupancy or point mutation, resulted in the ability to disrupt these oligomeric structures in living cells. However, within the D1-D2 heterooligomer, conformational alteration by antagonist occupancy of one receptor was unable to affect the conformation of the other, and we defined a structural alteration that prevented oligomerization of D1 and D2 receptors, indicating that the arrangement of the receptors within the heterooligomer may be substantially different from that within a homooligomer. Importantly, the strategy described provides a means of testing the robustness of the interaction between receptors, which will be useful to determine points of contact forming the oligomers. Fluorescent Proteins—cDNA sequences encoding GFP (27Prasher D.C. Eckenrode V.K. Ward W.W. Prendergast F.G. Cormier M.J. Gene (Amst.). 1992; 111: 229-233Crossref PubMed Scopus (1755) Google Scholar), pDsRed2 (28Matz M.V. Fradkov A.F. Labas Y.A. Savitsky A.P. Zaraisky A.G. Markelov M.L. Lukyanov S.A. Nat. Biotechnol. 1999; 17: 969-973Crossref PubMed Scopus (1507) Google Scholar), and pDsRed2-nuc were obtained from CLONTECH (Palo Alto, CA). Cell Culture—HEK cells grown on 60-mm plates in minimum essential medium were transfected with 0.5-2 μg of cDNA using Lipofectamine (Invitrogen). Dopamine antagonists (+)-butaclamol or SCH 23390, when used, was added to cells at 6, 22, 30, and 42 h, and cells were visualized by confocal microscopy at 48 h post-transfection. Microscopy—Live cells expressing GFP and pDsRed2 fusion proteins were visualized with an LSM510 Zeiss confocal laser microscope. In each experiment, 5-8 fields, containing 50-80 cells/field were evaluated, and the entire experiment was repeated 2-4 times (n = 3-5). Fluorocytometry—50,000 cells were added to each well (96-well plate) and transfected with 0.5 μg of cDNA. Minimum essential medium containing antagonists at varying concentrations were added to wells in pentuplicate. The drugs were prepared as 1 mm concentration stock and diluted in growth medium to achieve a concentration between 10 nm and 10 μm. After 48 h, cells were fixed with 4% paraformaldehyde and incubated with the primary antibody (rat anti-HA antibody, 1:200 dilution; Roche Applied Science) and secondary antibody conjugated to FITC (goat anti-rat antibody, 1:32 dilution; Sigma). Cell surface fluorescence was detected using a Cytofluor 4000 (PerSeptive Biosystems). Each experiment was repeated three or four times. Background fluorescence from media, cells, plastic, etc. was evaluated in each experiment and subtracted from the readings. DNA Constructs—All of the DNA encoding the GPCRs were from humans. Sequences encoding GPCRs were cloned into plasmid pEGFP, pDsRed2-N1, or pcDNA3. Receptor Constructs—The D1(S198A/S199A) and D1(S199A/S202A) receptors were prepared using the Quikchange mutagenesis kit (Stratagene) according to the manufacturer's instructions using the following sets of primers: D1(S198A/S199A), forward (5′-GGACATATGCCATGTCAGCCGCCGTAATAAGCTTTTACATCCC-3′) and reverse (5′-GGGATGTAAAAGCTTATTACGGCGGCTGAGATGGCATATGTCC-3′); D1(S199A/S202A), forward (5′-GCAGGACATATGCCATCTCATCCGCCGTAATAGCCTTTTACATCCCTGTGG-3′) and reverse (5′-CCACAGGGATGTAAAAGGCTATTACGGCGGATGAGATGGCATATGTCCTGC-3′). D1-NLS-GFP—Receptor DNA was subjected to PCR as previously reported (29Marchese A. George S.R. O'Dowd B.F. Lynch K. Identification and Expression of G Protein Coupled Receptors. Wiley-Liss, New York1998: 1-26Google Scholar). The reaction mixture consisted of H2O (32 μl), 10× Pfu buffer (Stratagene) (5 μl), dNTP (10 mm, 5 μl), Me2SO (5 μl), oligonucleotide primers (100 ng, 1 μl each), DNA template (100 ng), and Pfu enzyme (5 units). Total volume was 50 μl. PCR conditions were as follows: one cycle at 94 °C for 2 min and 30-35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min per cycle and then one cycle at 72 °C for 5 min. Primer Set for Amplification of the DNA Encoding the D1 Receptor—Primers were as follows: HD1-P1, 5′-GAGGACTCTGAACACCGAATTCGCCGCCATGGACGGGACTGGGCTGGTG-3′; HD1-P2, 5′-GTGTGGCAGGATTCATCTGGGTACCGCGGTTGGGTGCTGACCGTT-3′. The restriction site EcoRI was incorporated in the primer HD1-P1, and KpnI was incorporated into HD1-P2. The PCR product, containing no stop codon was subcloned into vector pEGFP at EcoRI and KpnI and in frame with the start codon of GFP. The NLS sequence, KKFKR, was inserted into DNA encoding the base of TM7 (helix 8) of the D1 dopamine receptor by PCR, replacing the sequence 336DFRKA. Primer Set for the Construction of DNA Encoding D1-NLS—Primers were as follows: HD1-NLSF, 5′-CCTAAGAGGGTTGAAAATCTTTTAAATTTTTTAGCATTAAAGGCATAAATG-3′; HD1-NLSR, 5′-GCCTTTAATGCTAAAAAATTTAAAAGATTTTCAACCCTCTTAGGATGC-3′. Using the DNA encoding D1-GFP as template, PCR with the primers HD1-P1 and HD1-NLSF resulted in a product of 1000 bp (PCR1). Using DNA encoding D1-GFP, PCR with primers HD1-P2 and HD1-NLSR resulted in a product of 300 bp (PCR2). A subsequent PCR carried out with HD1-P1 and HD1-P2 primers resulted in a product of 1300 bp using the product from PCR1 and the product from PCR2 as templates. The resulting DNA encoding D1-NLS was subcloned into vector pEGFP at EcoRI and KpnI restriction sites. All of the additional constructs described below were made using the same PCR method and experimental conditions as described above for the D1 dopamine receptor, but with the specific primers as described below. Primer Set for the Construction of D1-IC1-NLS—Primers were as follows: D1-NLSF-IC1, 5′-GTGCTGCCGTTAAAAAGTTCAAACGCCTGCGGTCCAAGG-3′; D1-NLSR-IC1, 5′-GGACCGCAGGCGTTTGAACTTTTTAACGGCAGCACAGACC-3′. KKFKR was inserted into the intracellular loop, replacing 49IRFRH. Primer Set for the Construction of D1-IC2-NLS—Primers were as follows: D1NLSF-IC2, 5′-CCGGTATGAGAAAAAGTTTAAACGCAAGGCAGCCTTC-3′; D1-NLSR-IC2, 5′-GGCTGCCTTGCGTTTAAACTTTTTCTCATACCGGAAAGG-3′. KKFKR was inserted into IC2, replacing 133RKMTP. Primer Set for the Construction of D1-IC3-NLS—Primers were as follows: D1NLSF-IC3, 5′-GGAAAGTTCTTTTAAGAAGAAGTTCAAAAGAGAAAC-3′; D1-NLSR-IC3, 5′-GTTTCTCTTTTGAACTTCTTCTTAAAAGAACTTTCC-3′. KKFKR was inserted into the IC3 segment of the D1 receptor, replacing 262MSFKR. Primer Set for D2-NLS-GFP—Primers were as follows: HDNLSF, 5′-CACCACCTTCAACAAAAAATTCAAAAGAGCCTTCCTGAAGATCC-3′; HD2-NLSR, 5′-GGATCTTCAGGAAGGCTCTTTTGAATTTTTTGTTGAAGGTGGTG-3′. The sequence KKFKR was inserted in the D2 receptor, replacing 431IEFRK. Primer Set for the Construction of SPGFP-D2—The D2 cDNA was isolated by the PCR method using the following set of primers: D2sp-BsrGI, 5′-TGTACAGCCGCCATGGATCCACTGAATCTGTCC-3′; D2sp-NotI, 5′-GAGTCGCGGCCGCTTCAGCAGTGGAGGATCTTCAGGAAGG-3′. This PCR product was then subcloned into the SP-GFP vector at restriction sites BsrGI and NotI. Primer Set for the Construction of SPGFP-D2-NLS—Using the SPGFP-D2 as template, the NLS KKFKR was introduced into helix 8 of the D2 by the PCR method using the following set of primers: HD2-NLSF, 5′-CACCACCTTCAACAAAAAATTCAAAAGAGCCTTCCTGAAGATCC-3′; HD2-NLSR, 5′-GGATCTTCAGGAAGGCTCTTTTGAATTTTTTGTTGAAGGTGGTG-3′. The sequence KKFRK was inserted in the D2 receptor, replacing 431IEFRK. Primer Set for the Construction of D5-GFP—The human D5 was isolated by PCR with primers HD5-EcoRI (5′ CTGGAATTCTGCAGATTCCAGCCCGAAATGCTGCCGCC-3′) and HD5-Kpn (5′-CGCCAGTGTGATGGATAATGGTACCGCATGGAATCCATTCGGGGTG-3′) and subcloned into the enhanced green fluorescent protein vector at the restriction sites EcoRI and KpnI. Primer Set for the Construction of SPGFP-D5—The human D5 was isolated by PCR with the following set of primers: D5-BsrGI, 5′-CCAGCCCGTGTACAAATGCTGCCGCCAGGCAGC-3′; D5-NotI, 5′-GCGGCCGCTTAATGGAATCCATTCGGGG-3′. This PCR product was then subcloned into the SP-GFP vector at restriction sites BsrGI and NotI. The Construction of D1-mRFP—mRFP1 in the pRSETb vector was a gift from Dr. Irine Prastio (Howard Hughes Medical Institute, University of California, San Diego). Using this vector as template, the mRFP was isolated by PCR using the following two primers: mRFP-BAMH, 5′-GATAAGGATCCGATGGCCTCCTCCGAGG-3′; mRFP-NOT, 5′-CGAATTCGCGGCCGCTAGGCGCCGGTGGAGTGGCGG-3′. This PCR product was then used to replace the GFP from the pEGFP-N1 vector at the restriction sites BamHI and NotI, thus creating the mRFP vector. Human D1 was excised from the D1-GFP construct by restriction digest with EcoRI and KpnI and subcloned into the mRFP vector at the same restriction sites EcoRI and KpnI and thus in frame with the mRFP. The Construction of D1-NLS (Helix 8)-mRFP—Human D1-NLS (helix 8) was excised from the D1-NLS (helix 8)-GFP construct by restriction digest with EcoRI and KpnI and subcloned into the mRFP vector at the same restriction sites EcoRI and KpnI and in frame with the mRFP. Primer Set for CysLT2-NLS-GFP—Primers used were LT2-NLSF, 5′-GCTGGGAAAAAATTTAAAAGAAGACTAAAGTCTGCAC-3′ and LT2-NLSR (5′-GTCTTCTTTTAAATTTTTTCCCAGCAAAGTAATAGAGC-3′). The sequence KKFKR was inserted into the cysteinyl leukotriene 2 receptor, replacing 310ENFKD. The initial objective was to generate a GPCR that would traffic to the nucleus under basal conditions and permit ligand-occupied conformational changes to modulate this process. Optimization of the Position of the NLS within the D1 Dopamine Receptor in a Conformation-dependent Site—To determine the optimal site for NLS incorporation that would provide the most efficient receptor translocation to the nucleus, the D1 dopamine receptor was modified. Since the proteins that carry NLS-containing proteins to the nucleus, such as the importins, are cytoplasmic, the optimal placement of the NLS was investigated by its introduction into various positions within the three intracellular loops, helix 8, and the carboxyl tail of D1; each of these locations is illustrated in Fig. 1b. When expressed in cells, the unmodified D1 receptor tagged with GFP was localized on the cell surface in the majority of the cells (>90%) at 48 h post-transfection as visualized by confocal microscopy (Fig. 2a, upper left panel). Cells transfected with the D1 receptor containing the NLS in helix 8 (D1-NLS) revealed a basal localization of receptor in the nucleus at 48 h post-transfection (Fig. 2a, upper right panel) observed in over 90% of cells. The localization of the receptor in the nucleus was confirmed using a nuclear dye (Hoechst 33342) (Fig. 2a, lower panels). Thus, incorporation of a NLS into the D1 receptor sequence in helix 8 resulted in efficient trafficking of the receptor under basal conditions to the nucleus. A series of tomographic images (Z-stacks) obtained by confocal microscopy confirmed the nuclear localization of the D1-NLS. Investigation of the effect of the NLS inserted in various other intracellular positions of the receptor revealed that D1 with an NLS inserted in the first intracellular cytoplasmic loop (D1-IC1-NLS) was expressed and detected in the nucleus in 85% of cells (Fig. 2b, left). D1 with the NLS inserted in the second intracellular cytoplasmic loop (D1-IC2-NLS) was expressed and detected in the nucleus in 51% of cells. In this case, over 40% of cells still had receptor detectable on the cell surface, indicating that incorporation of the NLS in this position was not as efficient in translocating the receptor from the cell surface. D1 with the NLS inserted in the third intracellular cytoplasmic loop (D1-IC3-NLS) was expressed and detected in the nucleus of 85% of cells (Fig. 2b, middle). D1 with an NLS inserted in a distal position in the carboxyl tail (D1-CT-NLS) was expressed and was detected at the cell surface and also in the nucleus (Fig. 2b, right). The distribution of D1-GFP-NLS where the NLS was attached distal to the GFP fused to D1, showed expression in the cytoplasm and cell surface but little expression in the nucleus (data not shown). To determine the effect of occupancy of the binding pocket on the NLS within various positions of the receptor on trafficking to the nucleus, we used several antagonists with inverse agonist properties to induce a conformational change in the receptor. Although receptor agonists would also induce conformational changes in the receptor, we wished to preclude agonist-induced internalization mechanisms, which would confound the evaluation of NLS-mediated trafficking. Cells expressing D1-NLS were treated with the dopamine receptor antagonist (+)-butaclamol ((+)BTC) or SCH 23390 6 h post-transfection. With (+)BTC (1 μm) treatment for 48 h, there was a very efficient retention of the D1-NLS receptor on the cell surface (85% of cells) with little translocation to the nucleus (Fig. 2c, left). This effect was also found with the D1 receptor-selective antagonist SCH 23390. Stereoselectivity of this effect was demonstrated by the lack of effect of (-)BTC (1 μm) treatment of the cells. In addition, the specificity of the D1 antagonist on receptor translocation was tested by treating D1-NLS-expressing cells with the D2 receptor-selective antagonist raclopride (1 μm), which was unable to retain the receptor at the cell surface. D1-NLS was expressed and treated with (+)BTC (500 nm) for 48 h, and at this time, the receptor was located at the cell surface (100% of cells). The (+)BTC was removed, and the receptor distribution was examined at 3, 6, 13, 16, 19, and 24 h. Between 13 and 16 h, the receptor had left the cell surface and was distributed to the nucleus in 80% of cells. Cells expressing D1-IC1-NLS treated with either (+)BTC (1 μm) or SCH 23390 (1 μm) also revealed retention of receptor at the cell surface in 82% of cells (Fig. 2c, middle) and 77% of the cells, respectively, compared with 76% of cells with receptor expression in the nucleus with no treatment. In contrast, cells expressing D1-IC3-NLS treated with antagonist (+)BTC or SCH 23390 revealed ∼90 and 84% of cells with receptor in the nucleus, indicating no ability of the drugs to retard the translocation of
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