Characterization of [formula omitted] binding to dopamine D 2-like receptors expressed in cell lines

Abstract

Recently, [ 125I]S(−)5- OH-PIPAT[5- hydroxy-2-(N- n-propyl-N-3′- iodo-2′- propenyl)aminotetralin] , a derivative of S(−)5-OH-DPAT(5-hydroxy- N, N-di-n-propyl-aminotetralin), was reported to be a better radioiodinated dopamine D 2-like receptor ligand than the previously reported iodinated ligand, [ 125I]R(+)7- OH-PIPAT . Therefore, in the present study, the binding profile of [ 125I]S(−)5- OH-PIPAT to D 2-like receptors expressed in cell lines was established. High binding affinity ( K d = 0.3–0.4 nM) and NaCl sensitivity were displayed with this ligand in membranes of human embryonic kidney (HEK293) cells expressing either human D 2 or rat D 3 receptors and in Chinese hamster ovary (CHO) cells expressing human dopamine D 4 receptors. Specific binding to D 2 and D 4 receptors was significantly increased in the presence of 2 mM MgCl 2 and decreased in the presence of 100 μM 5′-guanylyl-imidodiphosphate (GMP-PNP). This finding is consistent with reports that 2-aminotetralin compounds display agonist properties. The specific binding to D 3 receptors however, was not affected by either MgCl 2 or GMP-PNP. This lack of GMP-PNP sensitivity for D 3 receptors may result from inadequate G protein-receptor coupling in this cell line. The rank order of potency for inhibition of [ 125I]S(−)5- OH-PIPAT binding with various dopamine agents was consistent with reported values for D 2, D 3 and D 4 receptors. In membranes prepared from Spodoptera frugiperda (Sf9) cells infected with baculovirus that contains DNA encoding D 3 receptors, [ 125I]S(−)5- OH-PIPAT recognized only 70% of the receptor population labeled by [ 125I]NCQ298. This new ligand offers several unique advantages, including high specific activity, high binding affinity and selectivity for D 2-like receptors, that make it an excellent probe for the investigation and the characterization of dopamine D 2-like receptors.