Delineation of the Structural Basis for the Activation Properties of the Dopamine D1 Receptor Subtypes
1999; Elsevier BV; Volume: 274; Issue: 45 Linguagem: Inglês
10.1074/jbc.274.45.31882
ISSN1083-351X
AutoresRafal Iwasiow, Marie‐France Nantel, Mario Tiberi,
Tópico(s)Neurotransmitter Receptor Influence on Behavior
ResumoTo delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wild-type D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes. To delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wild-type D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes. G protein-coupled receptor terminal receptor locus transmembrane third extracellular loop human embryonic kidney 293 cells The classical paradigm for G protein-coupled receptor (GPCR)1 activation is described by the binding of an agonist to an inactive receptor state (R). This process leads to the formation of an active receptor state (R*) which interacts with heterotrimeric GTP-binding proteins (G proteins) to initiate a variety of intracellular signaling events. In recent years, however, mutagenesis studies have led to a notion asserting that GPCRs exist in an equilibrium between two interchangeable conformational states, R and R* (1Samama P. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar, 2Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar, 3Leff P. Trends Pharmacol. Sci. 1995; 16: 89-97Abstract Full Text PDF PubMed Scopus (474) Google Scholar). In the absence of ligand (agonist), GPCRs are predominantly maintained in an inactive R state by intramolecular constraints that prohibit the interaction with G proteins. These intramolecular constraints are released upon agonist binding or by mutations. Indeed, mutations in the carboxyl end of the third cytoplasmic loop of GPCRs can result in mutant receptors displaying high levels of agonist independent activity or constitutive activation (1Samama P. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar, 4Cotecchia S. Exum S. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2896-2900Crossref PubMed Scopus (286) Google Scholar, 5Kjelsberg M.A. Cotecchia S. Ostrowski J. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 1430-1433Abstract Full Text PDF PubMed Google Scholar, 6Ren Q. Kurose H. Lefkowitz R.J. Cotecchia S. J. Biol. Chem. 1993; 268: 16483-16487Abstract Full Text PDF PubMed Google Scholar). Constitutively active GPCRs have a greater propensity to adopt an R* state in the absence of agonists (1Samama P. Cotecchia S. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar, 2Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar). Most importantly, naturally occurring activating mutations in GPCRs have been shown to underlie various pathological conditions (2Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar, 7Vassart G. Horm. Res. 1997; 48: 47-50Crossref PubMed Scopus (21) Google Scholar). In addition, recent studies have shown that differences in the degree of constitutive activation may underlie the basis for GPCR multiplicity recognizing the same natural ligand (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar, 9Hasegawa H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 1857-1860Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). For instance, the dopaminergic D1 receptor subtypes have been demonstrated to display different levels of agonist-independent activity (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar, 10Charpentier S. Jarvie K.R. Severynse D.M. Caron M.G. Tiberi M. J. Biol. Chem. 1996; 271: 28071-28076Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 11Cardinaud B. Sugamori K.S. Coudouel S. Vincent J.-D. Niznik H.B. Vernier P. J. Biol. Chem. 1997; 272: 2778-2787Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Dopamine elicits its broad physiological effects through the interaction with specific classes of GPCRs: the D1-like and D2-like receptors (12Missale C. Nash S.R. Robinson S.W. Jaber M. Caron M.G. Physiol. Rev. 1998; 78: 189-225Crossref PubMed Scopus (2773) Google Scholar). In mammals, the dopaminergic D1-like receptors are divided into two subtypes referred to as D1A (or D1) and D1B (or D5), respectively (12Missale C. Nash S.R. Robinson S.W. Jaber M. Caron M.G. Physiol. Rev. 1998; 78: 189-225Crossref PubMed Scopus (2773) Google Scholar). The D1A and D1B receptor subtypes couple to the activation of adenylyl cyclase. The D1B receptor distinguishes itself from the D1A subtype by a higher constitutive activity (agonist-independent activity), an increased affinity and potency for agonists as well as a lower affinity for antagonist drugs (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar). These functional characteristics of the D1B receptor are highly reminiscent of those reported for constitutively active mutant GPCRs (2Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar). The molecular basis underlying the differences in the ligand binding and activation properties between the D1A and D1B receptor are little understood. In a recent study, we have described that replacement of a variant amino acid found in the carboxyl end of the third cytoplasmic loop of the D1A by the one found in the D1B receptor can induce partially the constitutive activation of the D1A receptor (10Charpentier S. Jarvie K.R. Severynse D.M. Caron M.G. Tiberi M. J. Biol. Chem. 1996; 271: 28071-28076Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In an opposite fashion, a mutant D1B receptor harboring the variant D1A amino acid exhibits a decreased level of constitutive activity as well as the binding and coupling properties similar to those of the wild-type D1A receptor (10Charpentier S. Jarvie K.R. Severynse D.M. Caron M.G. Tiberi M. J. Biol. Chem. 1996; 271: 28071-28076Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). These mutant receptors display no modification in their ability to interact with antagonists. Overall, these results suggest that the carboxyl end of the third cytoplasmic loop plays a role in constraining the D1A and D1B receptor into their inactive and active allosteric states, respectively. However, the results also indicate that the molecular properties of these two D1 receptor subtypes can only be explained partially by amino acid sequences of the carboxyl end of the third cytoplasmic loop. Therefore, it is likely that other structural determinants within these receptors exist to define the intramolecular interactions responsible for the distinct features of the D1A and D1B receptors. Indeed, studies have shown that mutations occurring in transmembrane regions, extracellular loops, or the cytoplasmic tail of GPCRs can lead to a constitutive activation (2Lefkowitz R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar, 9Hasegawa H. Negishi M. Ichikawa A. J. Biol. Chem. 1996; 271: 1857-1860Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar,13Parker E.M. Ross E.M. J. Biol. Chem. 1991; 266: 9987-9996Abstract Full Text PDF PubMed Google Scholar, 14Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. EMBO J. 1996; 15: 3566-3578Crossref PubMed Scopus (361) Google Scholar, 15Scheer A. Fanelli F. Costa T. De Benedetti P.G. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (200) Google Scholar, 16Nanevicz T. Wang L. Chen M. Ishii M. Coughlin S.R. J. Biol. Chem. 1996; 271: 702-706Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In the present study, we use a chimeric receptor approach (Fig. 1) to delineate further the potential structural determinants that underlie the molecular properties of the human D1A and D1B receptors. We report that chimeric D1A/D1B receptors harboring the terminal receptor locus (TRL) cassette which includes the transmembrane region (TM) 6 and 7, the third extracellular loop (EL3), and the cytoplasmic tail display a constitutive activity, dopamine affinity, and potency that are indistinguishable from their respective cognate wild-type receptors. Furthermore, studies with chimeric D1A/D1B receptors containing only the EL3 region suggest an important role for this region in the agonist-mediated maximal activation (intrinsic efficacy) of the D1 receptor subtypes. The present study identifies an important structural domain regulating the activation process of the D1A and D1B receptor but demonstrates also that the molecular determinants involved in the GPCR activation properties (constitutive activation, agonist potency, and intrinsic efficacy) can be separated. N-[methyl-3H]SCH23390 (84 Ci/mmol), [3H]adenine (24 Ci/mmol), and [14C]cAMP (275 mCi/mmol) were from Amersham Pharmacia Biotech. Dopamine, deschloro-SCH23390 (SCH23982), flupentixol, and (+)-butaclamol were purchased from Research Biochemicals International. 1-Methyl-3-isobutylxanthine was obtained from Sigma. To construct the chimeric receptors, we took advantage of the high degree of nucleotide identity between the human D1A and D1B receptor (17Jarvie R.K. Caron M.G. Adv. Neurol. 1993; 60: 325-333PubMed Google Scholar). Using the conserved BclI restriction site located within the nucleotide sequence coding for the TM6, we constructed two chimeric D1A and D1B receptors harboring the TRL cassette of their respective cognate wild-type counterparts (Fig. 1). The TRL cassette includes sequences coding for the TM6 and TM7 as well as the EL3 and carboxyl cytoplasmic tail. Moreover, the EL3 region of the D1A and D1B receptor was exchanged to create two additional chimeric receptors. The swapping of the EL3 region was done by gene splicing using a polymerase chain reaction-based overlap extension approach. The chimeric constructs were subcloned in pBluescript II SK+ (Stratagene) and the identity of the chimeras confirmed by dideoxy sequencing using Sequenase version 2.0 kit (U. S. Biochemical Corp.). Expression constructs for the wild-type and chimeric D1A and D1B receptors were engineered into the expression vector pCMV5. Human embryonic kidney 293 (HEK293) cells were from American Type Culture Collection (Manassas, VA). HEK293 cells were cultured at 37 °C and 5% CO2 in minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum and gentamicin (100 μg/ml) (Life Technologies, Inc.). Cells were seeded into 100-mm dishes (2.5 × 106cells/dish) and transiently transfected with 0.25–5 μg of DNA/dish using a modified calcium phosphate precipitation procedure as described (18Didsbury J.R. Uhing R.J. Tomhave E. Gerard C. Gerard N. Snyderman R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11564-11568Crossref PubMed Scopus (129) Google Scholar). After an overnight incubation with the DNA-calcium phosphate precipitate, HEK293 cells were washed with phosphate-buffered saline, trypsinized, reseeded in 150-mm dishes and grown for an additional 36–40 h. Transfected HEK293 cells were then washed with cold phosphate-buffered saline, scraped in ice-cold lysis buffer (10 mm Tris-HCl, pH 7.4, 5 mm EDTA), and centrifuged twice at 40,000 × g for 20 min at 4 °C. The pellet was resuspended in lysis buffer using a Brinkmann Polytron (17,000 r.p.m. for 15 s). The crude membranes were frozen in liquid nitrogen and stored at −80 °C until used. Frozen membranes were thawed on ice and resuspended in binding buffer (50 mm Tris-HCl, pH 7.4, 120 mm NaCl, 5 mm KCl, 4 mmMgCl2, 1.5 mm CaCl2, 1 mm EDTA) using a Brinkmann Polytron. Binding assays were performed with 100 μl of membranes in a total volume of 500 μl using N-[methyl-3H]SCH23390 as radioligand. Saturation studies were done using concentrations ofN-[methyl-3H]SCH23390 ranging from 0.01 to 6 nm. Nonspecific binding was delineated using 10 μm flupentixol. For competition studies, membranes were incubated with a constant concentration ofN-[methyl-3H]SCH23390 (∼0.6 nm) and increasing concentrations of competing ligand. Competition studies using dopamine were done in the presence of 0.1 mm ascorbic acid. Binding assays were incubated for 90 min at room temperature and terminated using rapid filtration through glass fiber filters (GF/C, Whatman). The filters were washed three times with 5 ml of cold washing buffer (50 mm Tris-HCl, pH 7.4, 120 mm NaCl) and the bound radioactivity was determined by liquid scintillation counting (Beckman Counter, LS1701). Protein concentration was measured using the Bio-Rad assay kit with bovine serum albumin as standard. To determine the equilibrium dissociation constant (Kd) and binding capacity (R) values, binding isotherms were analyzed using the nonlinear curve-fitting program LIGAND (19Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7772) Google Scholar). Regulation of adenylyl cyclase activity by wild-type and chimeric D1A and D1B receptors was assessed using a whole cell cAMP assay as described previously (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar). Following overnight incubation with the DNA-calcium phosphate precipitate, HEK293 cells were reseeded in 6- or 12-well dishes. The next day, HEK293 cells were cultured in fresh minimal essential medium containing 5% (v/v) fetal bovine serum, gentamicin (100 μg/ml), and [3H]adenine (2 μCi/ml; 24 Ci/mmol) for 18–24 h at 37 °C and 5% CO2. The labeling medium was then removed and HEK293 cells incubated in 20 mm HEPES-buffered minimal essential medium containing 1 mm1-methyl-3-isobutylxanthine in the presence or absence of dopamine for 30 min at 37 °C (in the presence of 0.1 mm ascorbic acid). At the end of the incubation period, the medium was aspirated, and each well filled with 1 ml of lysis solution containing 2.5% (v/v) perchloric acid, 1 mm cAMP, and [14C]cAMP (2.5–5 nCi, ∼5,000–10,000 cpm) for 20–30 min at 4 °C. The lysates were then transferred to tubes containing 0.1 ml of 4.2m KOH (neutralizing solution), and precipitates were sedimented by a low-speed centrifugation (1,500 rpm) at 4 °C. The amount of intracellular [3H]cAMP was determined from supernatants purified by sequential chromatography using Dowex and alumina columns as described before (20Johnson R.A. Salomon Y. Methods Enzymol. 1991; 195: 3-21Crossref PubMed Scopus (122) Google Scholar). The amount of [3H]cAMP (CA) over the total amount of intracellular [3H]adenine (TU) was calculated to determine the relative adenylyl cyclase activity (CA/TU). Dose-response curves to dopamine were analyzed by a four-parameter logistic equation using ALLFIT (21DeLéan A. Munson P.J. Rodbard D. Am. J. Physiol. 1978; 235: E97-E102Crossref PubMed Google Scholar). Receptor expression was determined using a saturating concentration (∼6 nm) ofN-[methyl-3H]SCH23390. Equilibrium dissociation binding constants (Kd) are expressed using the geometric mean ± S.E. All other data are reported as arithmetic means ± S.E. All statistical tests used in the present study have been described elsewhere (22Sokal R.R. Rohlf F.J. Biometry. 2nd Ed. W. H. Freeman and Company, New York1981Google Scholar, 23Motulsky H. Intuitive Biostatistics. Oxford University Press, New York1995Google Scholar). Prior to the statistical treatment of data, the homoscedasticity (homogeneity of variances) was assessed using either the Bartlett or Hartley tests. Then, one-sample t test and analysis of variance (one-way ANOVA) were performed to determine the statistical significance of the data. To establish the statistical significance of differences between pairs of means, a posteriori comparisons were performed using either the Bonferroni test (nonheterogeneity of variances) or the Games and Howell method (heterogeneity of variances). The level of significance was established at p < 0.05. The binding affinities (Kd values) of the radioligandN-[methyl-3H]SCH23390 for wild-type and chimeric human D1 receptors obtained using saturation studies are summarized in Table I. Results indicate that the chimeric receptors retain their ability to bindN-[methyl-3H]SCH23390 with high affinity. In addition, no statistical differences between the binding capacities of wild-type and chimeric receptors were detected (8–10 pmol/mg of protein). These results suggest that swapping the TRL cassette between the two receptors does not alter significantly the protein folding necessary for appropriate cell surface expression.Table IDissociation constant (Kd) and binding capacity (R) values for binding of [N-[methyl-3 H]-SCH23390 to wild-type and chimeric receptorsKd and R values are expressed as geometric and arithmetic means, respectively. Means are from six to eight experiments done in duplicate determinations. Open table in a new tab Kd and R values are expressed as geometric and arithmetic means, respectively. Means are from six to eight experiments done in duplicate determinations. Competition studies were performed to determine whether the TRL contains the underlying structural requirements involved in the dopamine binding to wild-type human D1A and D1B receptors. Dopamine exhibits a higher affinity for the D1B subtype in comparison with the D1A receptor (Table II) as previously reported (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar, 10Charpentier S. Jarvie K.R. Severynse D.M. Caron M.G. Tiberi M. J. Biol. Chem. 1996; 271: 28071-28076Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The chimera 1 displays an affinity for dopamine which is highly reminiscent of the binding affinity observed for the wild-type D1B receptor (Table II). In an opposite fashion, the chimera 2 binds dopamine with an affinity very similar to the one measured for the wild-type D1A receptor (Table II). To strengthen further the dominant role the TRL cassette plays in determining the D1A and D1B receptor conformations responsible for the distinct dopamine binding affinity, we calculated the free binding energy using the relation ΔG = −RT ln (1/Kd) (24Catterall W.A. Science. 1989; 243: 236-237Crossref PubMed Scopus (2) Google Scholar). As depicted in Fig. 2 A, the calculated net free energy difference relative to the dopamine binding energy for the wild-type D1A receptor suggests that chimera 1 displays a reduction in the binding energy preference for dopamine. This reduction is statistically different from the wild-type D1A receptor but indistinguishable from the wild-type D1B receptor. Meanwhile, the binding energy preference of chimera 2 for dopamine is not statistically different from the wild-type D1A receptor. In addition, the binding energy preference of chimera 2 for dopamine exhibits an increase that is statistically different from the wild-type D1B and chimera 1 (Fig. 2 A).Table IIDissociation constants (Kd) for binding of dopaminergic ligands to wild-type and chimeric receptorsKd values are expressed as geometric mean ± S.E. of 15–18 experiments done in duplicate determinations. DA, dopamine; FLU, flupentixol; BUTA, (+)-butaclamol; SCH, SCH23982. Open table in a new tab Kd values are expressed as geometric mean ± S.E. of 15–18 experiments done in duplicate determinations. DA, dopamine; FLU, flupentixol; BUTA, (+)-butaclamol; SCH, SCH23982. Previous studies have shown that antagonists or antipsychotic drugs bind with a lower affinity to the D1B receptor in comparison with the D1A receptor (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar, 10Charpentier S. Jarvie K.R. Severynse D.M. Caron M.G. Tiberi M. J. Biol. Chem. 1996; 271: 28071-28076Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). We tested the binding affinity of flupentixol and (+)-butaclamol, two antipsychotic drugs having a distinct chemical structure and displaying inverse agonism at the D1A and D1B receptors (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar, 25Milligan G. Bond R.A. Lee M. Trends Pharmacol. Sci. 1995; 16: 10-13Abstract Full Text PDF PubMed Scopus (283) Google Scholar). As shown in Table II, both drugs have lower affinity for the wild-type D1B receptor as reported before (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar). The binding affinities of flupentixol for the chimera 1 and 2 were not statistically different from the wild-type D1A and D1B receptors, respectively (Table II, Fig. 2 B). Interestingly, the small fold difference in the flupentixol affinity (∼1.5-fold) observed between the wild-type D1 receptors remains unchanged with the exchange of the TRL cassette. In striking contrast to flupentixol, (+)-butaclamol displays a greater fold difference in the binding affinity (∼5-fold) between the two D1 receptor subtypes (Table II). As shown in Fig. 2 C, the wild-type D1B receptor has an increased binding energy preference for (+)-butaclamol in comparison with the wild-type D1A receptor. The chimera 1 binds (+)-butaclamol with an affinity which is not statistically different from the wild-type D1A receptor (Table II, Fig.2 C). However, our binding data indicate that chimera 2 has an increased affinity for (+)-butaclamol (Table II, Fig.2 C). Indeed, the net binding energy preference for (+)-butaclamol of the chimera 2 is decreased in comparison with the wild-type D1B receptor but remains statistically different from the wild-type D1A subtype. We then studied the binding properties of the benzazepine SCH23982 which is structurally different from both flupentixol and (+)-butaclamol. This benzazepine has been described as a classical antagonist that binds preferentially to D1-like receptors (26Niznik H.B. Mol. Cell. Endocrinol. 1987; 54: 1-22Crossref PubMed Scopus (84) Google Scholar). In the present study, SCH23982 exhibits lower affinity for the wild-type D1B subtype (Table II) which correlates with a significant increase in the binding energy preference while contrasted to the wild-type D1A receptor (Fig. 2 D). Surprisingly, chimera 1 and 2 bind to SCH23982 with an increased affinity that is statistically significant in comparison with the wild-type D1A or D1B receptor (Table II, Fig.2 D). This trend is also observed using the radiolabeled benzazepine analogN-[methyl-3H]SCH23390 (Table I). Previous studies have shown that the D1B receptor shares the functional features of constitutively activated mutant GPCRs (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar, 10Charpentier S. Jarvie K.R. Severynse D.M. Caron M.G. Tiberi M. J. Biol. Chem. 1996; 271: 28071-28076Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The role of the TRL cassette in the agonist-independent activation of adenylyl cyclase by wild-type and chimeric receptors was assessed using a whole cell cAMP assay. The results are summarized in Fig. 3. In brief, the D1B receptor has a 3.5-fold higher agonist independent activity than the D1A receptor as shown previously (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar, 10Charpentier S. Jarvie K.R. Severynse D.M. Caron M.G. Tiberi M. J. Biol. Chem. 1996; 271: 28071-28076Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Interestingly, chimera 1 shows an increase in its constitutive activation that is statistically different from the wild-type D1A but indistinguishable from the wild-type D1B receptor (Fig. 3). In striking contrast, chimera 2 exhibits a significant decrease of its agonist independent activity when compared with the wild-type D1B receptor (Fig. 3). In fact, the constitutive activation level of chimera 2 is highly reminiscent of the one measured for the wild-type D1A receptor. Differences in the agonist-mediated coupling properties of the D1A and D1B receptors have been described previously (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar). To test whether the TRL cassette delineates the structural requirements for the dopamine potency and intrinsic efficacy, dose-response curves were done in HEK293 cells transfected with the wild-type and chimeric receptors. As depicted in Fig.4 A, the dopamine potency is about 10-fold superior at the wild-type D1B receptor in comparison with the wild-type D1A, a value in agreement with previous studies (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar, 10Charpentier S. Jarvie K.R. Severynse D.M. Caron M.G. Tiberi M. J. Biol. Chem. 1996; 271: 28071-28076Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Chimera 1 exhibits an increase in dopamine potency as compared with its wild-type D1A counterpart (Fig. 4 A). The potency of dopamine at the chimera 1 is not statistically different from the wild-type D1B receptor. Alternatively, chimera 2 displays a loss of dopamine potency that is significantly different from the wild-type D1A and D1B receptor (Fig. 4 A). Fig. 4 B shows that the maximal stimulation elicited by the wild-type D1A receptor is significantly higher than the wild-type D1B receptors as described before (8Tiberi M. Caron M.G. J. Biol. Chem. 1994; 269: 27925-27931Abstract Full Text PDF PubMed Google Scholar). Interestingly, chimera 1 and 2 elicited a maximal activation of adenylyl cyclase that is identical to t
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