Vol. 295, Issue 1, 274-283, October 2000

G-Protein Sensitivity of Ligand Binding to Human Dopamine D₂ and D₃ Receptors Expressed in Escherichia coli: Clues for a Constrained D₃ Receptor Structure¹

Jurgen F. M. Vanhauwe² , Katty Josson, Walter H. M. L. Luyten, Arnold J. Driessen and Josée E. Leysen

Department of Biochemical Pharmacology (J.F.M.V., K.J., J.E.L.), and Department of Functional Genomics (W.H.M.L.L.), Janssen Research Foundation, Belgium; and Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands (A.J.D.)

Human dopamine D₂ and D₃ 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 [¹²⁵I]iodosulpride and [³H]7-OH-DPAT, were similar for hD₃ receptors in E. coli and CHO cell membranes. Both agonists and antagonists showed 2- to 25-fold lower binding affinities at hD₂ receptors in E. coli versus CHO cell membranes (measured with [³H]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 ([³H]7-OH-DPAT) binding at hD₂ receptors but not at hD₃ receptors. Also, [³H]7-OH-DPAT (nanomolar concentration range) binding was undetectable at hD₂ but clearly measurable at hD₃ receptors in E. coli membranes. Addition of a G_i/o-protein mix to E. coli membranes increased high affinity [³H]7-OH-DPAT binding in a concentration-dependent manner at hD₂ and hD₃ 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 hD₂ and hD₃ receptors. Thus, agonist binding to D₃ 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 D₃ receptors (versus D₂ receptors) is not due to its lower affinity for G-proteins but attributed to its lower capacity to activate these G-proteins.