Dopamine D2 receptors contain a cluster of serine residues in the fifth transmembrane domain that contribute to activation of the receptor as well as to the binding of agonists. We used rat D2S dopamine receptor mutants, each containing a serine-to-alanine substitution (S193A, S194A, S197A), to investigate the mechanism through which these residues affect activation of the receptor. Activation of the mutant receptor S194A was abolished in an agonist-dependent manner, such that dopamine no longer inhibited cAMP accumulation in C6 glioma cells or activated G protein-regulated K+ channels in Xenopus laevis oocytes, whereas the efficacy of several other agonists was unaffected. Dihydrexidine did not inhibit cAMP accumulation at either S193A or S194A. The decreased efficacy of dihydrexidine at S193A and S194A and dopamine at S194A was associated with a decreased ability to detect a GTP-sensitive high affinity binding state for these agonists. The ability of dopamine to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding via S194A also was decreased by approximately 50%. Finally, constitutive stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate binding and inhibition of adenylate cyclase by the D2S receptor was reduced by mutation of either S193 or S194. These data support the existence of multiple active receptor conformations that are differentially sensitive to mutation of serine residues in the fifth-transmembrane domain.
D 2L dopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D 2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D 2L receptors with the selective D 2 agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D 2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (G␣ i1 *, G␣ i2 *, G␣ i3 *, and G␣ o *) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTP␥S-stimulated adenylate cyclase, respectively. To assess the specificity of D 2L -G␣ coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein ␣ subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D 2L cells with HSV-G␣ o * rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-G␣ i1 *, HSV-G␣ i2 *, or HSV-G␣ i3 * failed to rescue either response. In summary, the current study provides strong evidence that the D 2L dopamine receptor couples to G␣ o in neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D 2 receptor activation on adenylate cyclase activity. Key words: dopamine D 2L receptors; G␣ i/o ; NS20Y neuroblastoma; adenylate cyclase; pertussis toxin; heterologous sensitizationAlterations in D 2 -like dopamine receptors and their signaling pathways are thought to be involved in the etiology and treatment of many neuropsychiatric disorders, including schizophrenia, depression, Parkinson's disease, and drug abuse. Hence, identifying the signaling pathways evoked after D 2 -like dopamine receptor activation may help us to understand the biochemical changes that occur in clinical settings. Such studies in native neuronal tissues are made difficult by the molecular diversity of D 2 -like dopamine receptors (D 2S , D 2L , D 3 , and D 4 ), the number of pertussis toxin (P TX)-sensitive G-proteins through which they can couple (G␣ i1 , G␣ i2 , G␣ i3 , G␣ oa , and G␣ ob ), and the many signal pathways modulated by D 2 -like receptor activation (Huff, 1997;Watts and Neve, 1997).One of the characteristic features of D 2 -like dopamine receptors and other G␣ i /o -coupled receptors is P TX-sensitive inhibition of cAM P accumulation. Also, persistent stimulation of G␣ i / o-coupled receptors such as the D 2 -like dopamine receptors and the opioid receptor result...
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.
The D4 dopamine receptor, a member of the D2-like dopamine receptor family, may be important in the etiology and treatment of schizophrenia. The present study was designed to examine the effects of dopamine agonist exposure on adenylate cyclase activity in HEK293 cells stably expressing recombinant-D4 receptors. Two hour pretreatment with dopamine receptor agonists resulted in heterologous sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells expressing the D4.2, D4.4, or D4.7 dopamine receptor variant. The potency and efficacy of dopamine for sensitization of cyclic AMP accumulation was comparable at all D4 receptor variants. D4 dopamine receptor-mediated sensitization was blocked by the D4 antagonist, clozapine, and prevented by overnight pretreatment with pertussis toxin, implying a role for Gi/Go proteins in heterologous sensitization. Further, long-term (18 h) agonist exposure resulted in a greater degree of sensitization of forskolin-stimulated cyclic AMP accumulation in both intact cells and membrane preparations of cells expressing the D4 receptor, compared to 2 h agonist exposure, without altering the density of the receptors. In addition, long-term agonist exposure decreased the abundance of Gialpha without altering the abundance of Gsalpha, whereas short-term agonist treatment had no effect on the immunoreactivity of either G protein. In summary, long-term agonist-induced sensitization of adenylate cyclase by the D4 receptor may involve mechanisms that do not contribute to short-term sensitization.
A model describing the binding of catecholamines to receptors has been developed largely on the basis of in nitro mutagenesis of the B,-adrenergic receptor. According to this model, three points of interaction contribute much of the binding energy of catecholamines. A conserved aspartic acid residue in the third transmembrane domain, Asp-1 13 of the B,-adrenergic receptor, participates in an ionic interaction with the charged amine of agonists and antagonists, whereas two of three conserved serine residues clustered in the fifth transmembrane domain, Ser-204 and Ser-207, form hydrogen bonds with the m-and p-hydroxyl groups of catecholamine agonists [l]. We set out to assess the validity of this model for the dopamine D2 receptor.Although, the D2 receptor has only -20% amino acid identity with the P,-adrenergic receptor, and the pharmacological profiles of the two receptors differ greatly, amino acid identity in the transmembrane repons that are thought to form the binding pocket is much higher (39%) [2]. Furthermore, dopamine is a weak agonist at the P2adrenergic receptor (K. A. Neve and B. L. Wiens, unpublished work), and P-receptor agonists have some efficacy at the D2 receptor [ 3 ] .
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