title: G-protein sensitivity of ligand binding to human dopamine D(2) and D(3) receptors expressed in Escherichia coli: clues for a constrained D(3) receptor structure.
abstract: Human dopamine D(2) and D(3) receptors were expressed in Chinese hamster ovary (CHO) and Escherichia coli cells to compare their ligand binding properties in the presence or absence of G-proteins and to analyze their ability to interact with G(i/o)-proteins. Binding affinities of agonists (dopamine, 7-OH-DPAT, PD128907, lisuride) and antagonists/inverse agonists (haloperidol, risperidone, domperidone, spiperone, raclopride, nemonapride), measured using [(125)I]iodosulpride and [(3)H]7-OH-DPAT, were similar for hD(3) receptors in E. coli and CHO cell membranes. Both agonists and antagonists showed 2- to 25-fold lower binding affinities at hD(2) receptors in E. coli versus CHO cell membranes (measured with [(3)H]spiperone), but the rank order of potencies remained similar. Purported inverse agonists did not display higher affinities for G-protein-free receptors. In CHO membranes, GppNHp decreased high affinity agonist ([(3)H]7-OH-DPAT) binding at hD(2) receptors but not at hD(3) receptors. Also, [(3)H]7-OH-DPAT (nanomolar concentration range) binding was undetectable at hD(2) but clearly measurable at hD(3) receptors in E. coli membranes. Addition of a G(i/o)-protein mix to E. coli membranes increased high affinity [(3)H]7-OH-DPAT binding in a concentration-dependent manner at hD(2) and hD(3) receptors; this effect was reversed by addition of GppNHp. The potency of the G(i/o)-protein mix to reconstitute high affinity binding was similar for hD(2) and hD(3) receptors. Thus, agonist binding to D(3) receptors is only slightly affected by G-protein uncoupling, pointing to a rigid receptor structure. Furthermore, we propose that the generally reported lower signaling capacity of D(3) receptors (versus D(2) receptors) is not due to its lower affinity for G-proteins but attributed to its lower capacity to activate these G-proteins.
authors.affiliation: Department of Biochemical Pharmacology, Janssen Research Foundation, Belgium.
authors.forename: J F
authors.initials: JF
authors.lastname: Vanhauwe
authors.forename: K
authors.initials: K
authors.lastname: Josson
authors.forename: W H
authors.initials: WH
authors.lastname: Luyten
authors.forename: A J
authors.initials: AJ
authors.lastname: Driessen
authors.forename: J E
authors.initials: JE
authors.lastname: Leysen
mesh_terms: D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000378:metabolism*; D050637:Receptors, Dopamine D3; D011994:Recombinant Proteins / Q000378:metabolism; D013764:Tetrahydronaphthalenes / Q000378:metabolism
chemical_list: C493593:DRD3 protein, human; D017448:Receptors, Dopamine D2; D050637:Receptors, Dopamine D3; D011994:Recombinant Proteins; D013764:Tetrahydronaphthalenes; D019204:GTP-Binding Proteins; C031349:7-hydroxy-2-N,N-dipropylaminotetralin