Delineation of the Structural Basis for the Activation Properties of the Dopamine D1 Receptor Subtypes

Abstract

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. 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 (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). mutations in GPCRs have to R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar, PubMed Scopus Google Scholar). In recent studies have that differences in the of constitutive activation the for the ligand Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, H. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the D1 receptor have to display levels of activity Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Dopamine the interaction with of the and receptors Caron M.G. PubMed Scopus Google Scholar). In the receptors are into two referred to as D1A and D1B Caron M.G. PubMed Scopus Google Scholar). The D1A and D1B receptor to the activation of adenylyl The D1B receptor from the D1A by a constitutive activity an increased affinity and for as well as a affinity for Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). These of the D1B receptor are highly reminiscent of for active mutant GPCRs R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar). The the differences in the ligand binding and activation properties between the D1A and D1B receptor are In a recent we have described that of a in the carboxyl end of the third cytoplasmic loop of the D1A by the in the D1B receptor can partially the constitutive activation of the D1A receptor S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In an a mutant D1B receptor harboring the D1A a of constitutive activity as well as the binding and coupling properties to of the wild-type D1A receptor S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These mutant receptors display in their to with Overall, suggest that the carboxyl end of the third cytoplasmic loop a role in the D1A and D1B receptor into their inactive and active states, respectively. However, the that the properties of two D1 receptor can partially by of the carboxyl end of the third cytoplasmic is that structural determinants receptors exist to the intramolecular interactions for the of the D1A and D1B receptors. Indeed, studies have that mutations in transmembrane extracellular or the cytoplasmic of GPCRs can to a constitutive activation R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar, H. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. Costa T. Cotecchia S. J. PubMed Scopus Google Scholar, A. Costa T. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the we a chimeric receptor 1) to delineate further the structural determinants that the properties of the human D1A and D1B receptors. that chimeric receptors harboring the terminal receptor locus cassette which the transmembrane region 6 and the third extracellular loop and the cytoplasmic display a constitutive dopamine and that are indistinguishable from their respective cognate wild-type receptors. Furthermore, studies with chimeric receptors containing the EL3 region suggest an role for region in the agonist-mediated maximal activation of the D1 receptor subtypes. 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PubMed Scopus Google Scholar). an with the cells were with in and for an additional cells were with in and for The in a for The were in and were and in binding 1 a were with of in a of as studies were from to 6 binding were with a and of studies dopamine were in the of were for and The were with of and the by the with as To the equilibrium and binding binding were the PubMed Scopus Google Scholar). of adenylyl cyclase activity by wild-type and chimeric D1A and D1B receptors a as described Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with the cells were in or The cells were in containing and for and The and cells in containing 1 in the or absence of dopamine for the of the end of the the and well with 1 of containing 1 and for The were to containing of and were by a The of intracellular from by and as described PubMed Scopus Google Scholar). The of the of intracellular to the adenylyl cyclase activity to dopamine were by a A. J. PubMed Google Scholar). a binding are the are as in the have described H. and Scholar, H. Scholar). to the of the of the or and of were to the of the To the of differences between of a were the of or the and of The of The binding of the for wild-type and chimeric human D1 receptors studies are in that the chimeric receptors their to with high In differences between the binding of wild-type and chimeric receptors were of These suggest that the TRL cassette between the two receptors the for and binding for binding of to wild-type and chimeric and R are as and respectively. are from to in in a and R are as and respectively. are from to in studies were to the TRL the structural involved in the dopamine binding to wild-type human D1A and D1B receptors. Dopamine a affinity for the D1B in with the D1A receptor as Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus 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 In an the chimera 2 binds dopamine with an affinity to the for the wild-type D1A receptor To further the role the TRL cassette in the D1A and D1B receptor for the dopamine binding we the binding the PubMed Scopus Google Scholar). in 2 the to the dopamine binding for the wild-type D1A receptor that chimera 1 displays a in the binding for This is from the wild-type D1A receptor indistinguishable from the wild-type D1B receptor. Meanwhile, the binding of chimera 2 for dopamine is from the wild-type D1A receptor. In the binding of chimera 2 for dopamine an that is from the wild-type D1B and chimera 1 2 for binding of to wild-type and chimeric are as of in in a are as of in studies have that or with a affinity to the D1B receptor in with the D1A receptor Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the binding affinity of and two a and displaying the D1A and D1B receptors Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Trends Pharmacol. Sci. 1995; 16: Full Text PDF PubMed Scopus Google Scholar). in have affinity for the wild-type D1B receptor as Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The binding of for the chimera 1 and 2 were from the wild-type D1A and D1B 2 the in the affinity observed between the wild-type D1 receptors unchanged with the exchange of the TRL In contrast to displays a greater in the binding affinity between the two D1 receptor in 2 the wild-type D1B receptor an increased binding for in with the wild-type D1A receptor. The chimera 1 binds with an affinity which is from the wild-type D1A receptor However, our binding that chimera 2 an increased affinity for Indeed, the binding for of the chimera 2 is in with the wild-type D1B receptor from the wild-type D1A the binding properties of the which is from and This described as a classical that binds to receptors PubMed Scopus Google Scholar). In the affinity for the wild-type D1B which with a in the binding to the wild-type D1A receptor 2 chimera 1 and 2 to with an increased affinity that is in with the wild-type D1A or D1B receptor This is observed the studies have that the D1B receptor the of mutant GPCRs Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The role of the TRL cassette in the activation of adenylyl cyclase by wild-type and chimeric receptors a The are in In the D1B receptor a agonist independent activity the D1A receptor as Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). chimera 1 an in constitutive activation that is from the wild-type D1A indistinguishable from the wild-type D1B receptor In chimera 2 a of agonist independent activity with the wild-type D1B receptor In the constitutive activation of chimera 2 is highly reminiscent of the for the wild-type D1A receptor. in the agonist-mediated coupling properties of the D1A and D1B receptors have described Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). To the TRL cassette the structural for the dopamine and were in cells with the wild-type and chimeric receptors. in the dopamine is the wild-type D1B receptor in with the wild-type a in with studies Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). 1 an in dopamine as with wild-type D1A The of dopamine the chimera 1 is from the wild-type D1B receptor. chimera 2 displays a of dopamine that is from the wild-type D1A and D1B receptor that the maximal elicited by the wild-type D1A receptor is the wild-type D1B receptors as described Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). chimera 1 and 2 elicited a maximal activation of adenylyl cyclase that is identical to their respective wild-type receptor counterparts suggest that differences the of the TRL cassette for the observed between the dopamine of the wild-type D1A and D1B receptors. the is that the structural determinants the D1A and maximal activation are the TRL our chimeric receptors. To we have engineered two additional chimeric D1A and D1B receptors that a domain of the TRL the EL3 that the low of between the EL3 of the D1A and D1B as in 1, suggest an structural role for region in the and activation of the D1 receptor subtypes. The are D1A and D1B receptors the EL3 region of their cognate wild-type were constructed a 1, In chimeric receptors display high levels of of binding studies that dopamine affinity for the hD1A-EL3B is increased with the wild-type D1A receptor In a chimeric D1B receptor the EL3 region of the D1A receptor a of dopamine These were in the binding for dopamine The binding affinity and binding for binding and dopamine to wild-type and chimeric EL3 and R are as and respectively. are from to in in a and R are as and respectively. are from to in The agonist independent activity of the hD1A-EL3B and hD1B-EL3A mutant receptors in cells expressing levels of receptors. The hD1B-EL3A mutant receptor displays a of agonist independent activity in with the wild-type D1B In the hD1A-EL3B chimeric receptor a gain in the of constitutive activation as with the wild-type D1A receptor. cells expressing receptor levels of wild-type or chimeric receptors were with a that a maximal activation of adenylyl described the wild-type D1A receptor a maximal activation of adenylyl cyclase the wild-type D1B receptor of cells expressing the hD1A-EL3B chimeric receptor with leads to a maximal activation of adenylyl cyclase that is indistinguishable from the maximal activation elicited by the wild-type D1B receptor Meanwhile, a full of the hD1B-EL3A mutant receptor or wild-type D1A receptor to a maximal activation of adenylyl cyclase In the we have a chimeric to the structural determinants involved in the activation properties of the D1 receptor subtypes. in to delineate or the binding and coupling of GPCRs Caron M.G. Lefkowitz R.J. PubMed Scopus Google Scholar, S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). have the that a low of between the TRL in the receptor of the In of the a of of between the D1A-TRL and D1B-TRL cassette low of the chimeric receptors their to high levels of in These suggest that the of the TRL structural the receptor The of our chimeric D1A and D1B receptors the agonist binding to the D1 receptor subtypes. the binding affinity of to the chimeric receptors unchanged in with their cognate wild-type receptors. displays a affinity for the D1B receptor in with the D1A receptor Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). These suggest that binding that are the TRL or is independent of the conformational the binding of to D1A and D1B receptor in the TRL of D1 receptor and upon interactions with the TRL Meanwhile, we show that binding of to the D1 receptor is by intramolecular Indeed, with chimera 2 a role for the D1A-TRL in to the wild-type D1A receptor to with a with a of the D1A-TRL the affinity of chimera 2, our that the structural determinants the TRL an role in the that the binding of In a exchange in the D1A receptor the D1B-TRL (chimera in the of a the affinity that the affinity of the D1B receptor for is independent of by the These the of D1 In the differences observed between the two suggest that exist to the active conformational state or that the receptor can adopt active Overall, our the notion that binding of to the D1A and D1B receptor structural with the the role of the TRL in the receptor of the receptor subtypes. described as a high affinity that binds to receptors PubMed Scopus Google Scholar). However, recent studies have that as a agonist the D1 a observed in cells Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google in and cells S. 1995; Scholar, M.A. P. J. PubMed Scopus Google Scholar, M.A. P. J. PubMed Scopus Google Scholar). This is studies that can in a agonist P. P. PubMed Scopus Google Scholar, Dopamine and and Scholar). chimeras a in the binding with their wild-type receptor counterparts 2 The reduced binding into a affinity of chimera 1 and 2 for is that the of in chimeric receptors that our that the conformational constraints of the chimeras the receptor affinity for by the of for the interactions with the In our suggest a role for TRL in interactions with receptor regions to the D1A and D1B receptor into a binding state for the of a between in the TRL and receptor regions with the intracellular that the two D1 receptor into a binding for The a structural domain herein to as that is fundamental in the and activation process of the human D1A and D1B receptor. that the TRL is involved in the intramolecular interactions that the binding and coupling properties of the D1 receptor subtypes. ligand binding and G coupling with chimera 2 suggest that the intramolecular interactions by the D1A-TRL predominantly the chimeric receptor in a as by a binding affinity and for and a the properties of chimera 2 are from of the wild-type D1A receptor. In the intramolecular interactions by the D1B-TRL chimera 1 to adopt a as by an increased binding affinity and for and a In chimera 1 constitutive activation properties that are indistinguishable from the wild-type D1B receptor. the D1B-TRL the structural determinants that the of active GPCRs R.J. Cotecchia S. Samama P. Costa T. Trends Pharmacol. Sci. 1993; 14: 303-307Abstract Full Text PDF PubMed Scopus (757) Google Scholar, Caron M.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). we S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the of a active can or with the In contrast to with mutations in the third cytoplasmic loop S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google a chimeric D1B receptor containing the D1A-TRL (chimera 2) displays the properties of a receptor with the wild-type D1B receptor. Overall, our suggest that the TRL cassette the to the D1A and D1B the properties of constitutive activation and agonist involved in the maximal activation of adenylyl Indeed, in cells and a of the D1B receptor or chimera 2 a maximal activation of adenylyl cyclase activity in with cells expressing the D1A receptor or chimera cells expressing the D1B receptor a for a receptor that the by the TRL the of structural However, is that the of receptor as our TRL the of regions which are in the of the activation properties of the D1 receptor subtypes. A of the of the TRL that a low of is the EL3 region we that the EL3 region involved in the activation process of the D1A and D1B receptor. In of a recent that the EL3 region is for the of agonist binding and independent activity of the receptor R.J. Pharmacol. PubMed Scopus Google Scholar). that the dopamine affinity with hD1A-EL3B and hD1B-EL3A mutant receptors partially the dopamine binding of their cognate wild-type receptor counterparts. The observed in dopamine affinity were in the agonist independent activation of hD1A-EL3B and to mutations in the region of the third intracellular loop S. Caron M.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we show that dopamine affinity and activity of the wild-type D1A and D1B receptors partially recapitulated with the EL3 loop However, and we that a of the EL3 region leads to a of the dopamine activation of adenylyl This is in marked contrast with our chimera 1 and EL3 an role in the intramolecular interactions involved in the maximal activation of adenylyl cyclase by the D1A and D1B to a in agonist binding and constitutive The of EL3 by differences in the of and In the and to of of the conformational of the D1A and D1B receptor. 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The of are of the for the maximal activation of adenylyl cyclase observed upon of the D1A and D1B receptor. and The of between D1A-TRL and D1B-TRL is the and Indeed, in the and the by two and respectively. studies have that the of receptors is an structural of agonist and binding whereas the coupling to proteins Caron M.G. Lefkowitz R.J. PubMed Scopus Google Scholar). the implicated in between for the human and receptors and affinity T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A role for the and in between the D1A and D1B receptor to However, studies chimeric receptor constructed from D1A and or D1A and and as structural determinants that of the properties of and receptors 1993; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, Pharmacol. PubMed Scopus Google Scholar, Pharmacol. 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Full Text Full Text PDF PubMed Scopus Google Scholar). the differences in the of the and of D1 receptors the of and the for their and receptor in the cytoplasmic an role in the of active and inactive of the D1A and D1B receptors. In the of between D1A-TRL and D1B-TRL is in the cytoplasmic of intracellular is that the cytoplasmic a role in the of and Meanwhile, studies have that the cytoplasmic can the formation of active and inactive receptor H. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). However, a recent that chimeric D1 receptors harboring cytoplasmic in the dopamine affinity and and constitutive activation M.A. P. J. PubMed Scopus Google Scholar). In our an receptor domain containing the structural determinants involved in the activation process of the human receptor subtypes. in our are to further the of the constitutive activation and G coupling of the D1A and D1B receptor. To our the the of mutations in GPCRs that can agonist constitutive activation, and activation of adenylyl for with the our to and for and the