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

Vanhauwe, J. F. M., Josson, K., Luyten, W. H. M. L., Driessen, A. J. & Leysen, J. E., 2000, In : Journal of Pharmacology and Experimental Therapeutics. 295, 1, p. 274-283 10 p.

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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 Gi/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 Gi/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 Gi/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.
Original languageEnglish
Pages (from-to)274-283
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Volume295
Issue number1
Publication statusPublished - 2000

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