Characterization of [I-125]S(-)5-OH-PIPAT binding to dopamine D-2-like receptors expressed in cell lines

Recently, [I-125]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, [I-125]R(+)7- OH-PIPAT. Therefore, in the present study, the binding profile of [I-125]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 MgCl2 and decreased in the presence of 100 mu M 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 MgCl2 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 [I-125]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, [I-125]S(-)5-OH-PIPAT recognized only 70% of the receptor population labeled by [I-125]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. (C) 1997 Elsevier Science Ltd.