Reciprocal Mutations in TM2/TM3 in a D2 Dopamine Receptor Background Confirms the Importance of this Microdomain as a Selective Determinant of Para‐Halogenated 1,4‐Disubstituted Aromatic Piperazines

Abstract

We recently demonstrated that in the D4 dopamine receptor the aromatic microdomain that spans the interface of the second and third transmembrane (TM) domains influences the high affinity interactions of extremely D4-selective ligands possessing a 1,4-disubstituted aromatic piperazine/piperidine (1,4-DAP) structure. On the basis of their substructural features and patterns of sensitivity to mutations constructed in a D4 receptor background, the D4-selective 1,4-DAPs were categorized as having two distinct modes of binding that we named mode-1 and mode-3 [1]. Here we extend these findings of the ligand-receptor structure-affinity relationships for some of these highly D4-selective 1,4-DAPs by measuring the effect of the corresponding reciprocal TM2/TM3 mutations constructed in a D2 dopamine receptor background on the binding affinity of the para-halogenated mode-1 ligands L750,667 and FAUC213. The results indicate that the D2-V2.61F + FV3.28-3.29LM mutant binds L750,667 and FAUC213 with significantly increased affinity, i.e., its binding profile becomes more D4-like. These findings further support the assignment of the TM2/TM3 aromatic microdomain encompassing positions 2.61 and 3.28-3.29 as a 1,4-DAP D4-selectivity microdomain and highlights the importance of the precise emplacement of aromatics in this microdomain as key to the selective molecular recognition of L750,667 and FAUC213.

Ligand Selectivity of D2Dopamine Receptors Is Modulated by Changes in Local Dynamics Produced by Sodium Binding

“…First, it explains the discrepancy (ϳ100-fold differences) in the reported affinities of L-745,870 and several other structurally similar D 4 -selective 1,4-DAPs for the D 2 -V2.61(91)F mutant and thus resolves an apparent contradiction in the literature (Simpson et al, 1999;Floresca et al, 2005). Second, it demonstrates how key molecular interactions between a ligand and a specific GPCR microdomain are influenced by occupancy of an allosteric site.…”

“…Although previous studies have revealed the affinity relationship between the discriminant structural features of 1,4-DAPs and the 1,4-DAP D 4 /D 2 selectivity-conferring positions 2.61(91), 3.28(110), and 3.29(111), the effects of mutations in this microdomain on the activity of 1,4-DAP ligands had not been described previously (Simpson et al, 1999;Schetz et al, 2000;Kortagere et al, 2004;Floresca et al, 2005). Therefore, because the D 2 -V2.61(91)F receptor exhibits sodium-sensitive affinity changes to 1,4-DAPs, we examined here the functional properties of L-745,870, L-750,667, and NGD 94-1 at the wild-type and D 2 -V2.61(91)F receptor.…”

“…Cells were then incubated the presence of the drug dilutions at 37°C for 20 min and then centrifuged at 1500g for 5 min. Drug dilutions (Simpson et al, 1999;Schetz et al, 2000;Kortagere et al, 2004;Floresca et al, 2005). Although most ligands bind an orthosteric binding site accessible from the extracellular face of the receptor (Floresca and Schetz, 2004), the sodium ion binds the receptor through an intracellular allosteric binding site formed by the interactions of transmembrane segments 2, 3, and 7 (Neve et al, 2001).…”

“…In an effort to determine the source of the discrepancy between the reported 97-fold increase in affinity for L-745,870 for an N-and C-terminally epitope-tagged human D 2 -V2.61(91)F mutant (Simpson et al, 1999) and the lack of change for the identical mutation in the rat receptor for the same ligand (Table 1) and several other ligands belonging to the same structural class (Table 1; Floresca et al, 2005), we systematically eliminated differences between the two experimental systems. Initially, we expressed our rat D 2 -V2.61(91)F mutant in the same HEK293 cell line used in the previous report.…”

“…In the process of determining the source of large discrepancies in binding affinity reported for 1,4-disubstituted aromatic piperidines/piperazines (1,4-DAPs) for the D 2 -V2.61(91)F mutant (Simpson et al, 1999;Floresca et al, 2005), we discovered a change, elicited by sodium binding, in the dynamic properties of the receptor that correlate with a dramatic increase in sodium sensitivity for both agonists and antagonists belonging to a similar structural class. This finding offers an opportunity to understand the allosteric mechanism of the effect produced by sodium binding.…”