We present the first model of dopamine D2 receptor transmembrane helices constructed directly from the bacteriorhodopsin (bR) coordinates derived from two-dimensional electron diffraction experiments. We have tested this model by its ability to accommodate rigid agonist and semirigid antagonist molecules which were docked into the putative binding pocket with stabilizing interactions. The model is consistent with structure-activity relationships of agonists and antagonists that interact with the receptor. It also illuminates data on a Na+ site for regulation of receptor function. The plausibility of the model is increased by its consistency with many mutagenesis studies on G protein-coupled receptors. Further, this model provides a basis to suggest testable molecular mechanisms for changes in the D2 conformational states for high- and low-affinity binding and signal transduction. Changes in the conformational state of the receptor are hypothesized to be due partly to movement of helix 7. In contrast to the model presented here, other published models were built using ideal helical structures or following the sense of the bacteriorhodopsin structure rather than the actual available coordinates. The presented model for the dopamine G protein-coupled receptor can be reconciled with the recent rhodopsin projection structure (Schertler, G. F. X.; Villa, C.; Henderson, R. Projection Structure of Rhodopsin.

A homology model of the dopamine D2 receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB ) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.

We have previously shown that using agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in three-dimensional quantitative structure-activity relationship studies (3D-QSAR) presents a unique opportunity for accuracy and precision in measurement. Thus, a comparison of affinity's structural determinants for a set of compounds at two different recombinant dopamine receptors represents an attainable goal for 3D-QSAR. A molecular database of bound conformations of 16 structurally diverse agonists was established by alignment with a high-affinity template compound for the D1 receptor, 3-allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4, 5-tetrahydro-1H-benzazepin. A second molecular database of the bound conformations of the same compounds was established against a second template for the D2 receptor, bromocriptine. These aligned structures suggested three-point pharmacophore maps (one cationic nitrogen and two electronegative centers) for the two dopamine receptors, which differed primarily in the height of the nitrogen above the plane of the catechol ring and in the nature of the hydrogen-bonding region. The ln(1/KL) values for the low-affinity agonist binding conformation at recombinant D1 and D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA (comparative molecular field analysis) of the 16 aligned structures. The resulting CoMFA models yielded cross-validated R2 (q2) values (standard error of prediction) of 0. 879 (1.471, with five principal components) and 0.834 (1.652, with five principal components) for D1 and D2 affinity, respectively. The simple R2 values (standard error of the estimate) were 0.994 (0.323) and 0.999 (0.116), respectively, for D1 and D2 receptor. F values were 341 and 2465 for D1 and D2 models, respectively, with 5 and 10 df. The predictive utility of the CoMFA model was evaluated at both receptors using the dopamine agonists, apomorphine and 7-OH-DPAT. Predictions of KL were accurate at both receptors. Flexible 3D searches of several chemical databases (NCI, MDDR, CMC, ACD, and Maybridge) were done using basic pharmacophore models at each receptor to determine the similarity of hit lists between the two models. The D1 and D2 models yielded different lists of lead compounds. Several of the lead compounds closely resembled high-affinity training set compounds. Finally, homology modeling of agonist binding to the D2 receptor revealed some consistencies and inconsistencies with the CoMFA-derived D2 model and provided a possible rationale for features of the D2 CoMFA contour map. Together these results suggest that CoMFA-homology based models may provide useful insights concerning differential agonist-receptor interactions at related receptors. The results also suggest that comparisons of CoMFA models for two structurally related receptors may be a fruitful approach for differential QSAR.

Keywords: receptors; G protein-coupled; phospholipid methylation; methionine; folic acid; membrane fluidity; purines; de novo synthesis Schizophrenia is a complex psychiatric disorder affecting approximately 1% of the population worldwide with a typical onset between the late teens and midthirties, often in the absence of significant prodromal psychiatric symptoms.1 Decades of research have yielded many theories on the origin of schizophrenia, although none provides a satisfying mechanistic explanation. Logically, dysfunctional neurotransmission is the most widely held theory, and specific abnormalities involving dopaminergic, 2 glutamatergic, 3 GABAergic 4 and nicotinic cholinergic 5 signaling have been proposed. Of these, the 'Dopamine Hypothesis' is dominant, based largely on the recognized therapeutic efficacy of drugs which block D2-like dopamine receptors (ie D2, D3 and D4 receptors), although their specific mechanism of benefit and the importance of individual receptor subtypes remains unclear. The D4

Agonist affinity changes dramatically as a result of serine to alanine mutations (S193A, S194A, and S197A) within the fifth transmembrane region of D2 dopamine receptors and other receptors for monoamine neurotransmitters. However, agonist 2D-structure does not predict which drugs will be sensitive to which point mutations. Modeling drug-receptor interactions at the 3D level offers considerably more promise in this regard. In particular, a comparison of the same test set of agonists across receptors differing minimally (point mutations) offers promise to enhance the understanding of the structural bases for drug-receptor interactions. We have previously shown that comparative molecular field analysis (CoMFA) can be applied to comparisons of affinity at recombinant D1 and D2 dopamine receptors for the same set of agonists, a differential QSAR. Here, we predicted agonist K(L) for the same set of agonists at wild type D2 vs S193A, S194A, and S197A receptors using CoMFA. Each model used bromocriptine as the template. ln(1/K(L)) values for the low-affinity agonist binding conformation at recombinant wild type and mutant D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA of the 16 aligned agonist structures. The resulting CoMFA models yielded cross-validated R(2) (q(2)) values ranging from 0.835 to 0.864 and simple R(2) values ranging from 0.999 to 1.000. Predictions of test compound affinities at WT and each mutant receptor were close to measured affinity values. This finding confirmed the predictive ability of the models and their differences from one another. The results strongly support the idea that CoMFA models of the same training set of compounds applied to WT vs mutant receptors can accurately predict differences in drug affinity at each. Furthermore, in a "proof of principle", two different templates were used to derive the CoMFA model for the WT and S193A mutant receptors. Pergolide was chosen as an alternate template because it showed a significant increase in affinity as a result of the S193A mutation. In this instance both the bromocriptine- and pergolide-based CoMFA models were similar to one another but different from those for the WT receptor using bromocriptine- or pergolide- as templates. The pergolide-based S193A model was more strikingly different from that of the WT receptor than was the bromocriptine-based S193A model. This suggests that a "dual-template" approach to differential CoMFA may have special value in elucidating key differences across related receptor types and in determining important elements of the drug-receptor interaction.