Investigation of the Role of Conserved Serine Residues in the Long Form of the Rat D₂ Dopamine Receptor Using Site‐Directed Mutagenesis

Abstract

Three serine residues (Ser193, Ser194, Ser197) in the fifth transmembrane‐spanning region of the D2 dopamine receptor have been mutated separately to alanine and the effects of the mutations determined in ligand‐binding experiments with [3H]spiperone. For many antagonists the mutations had little effect, showing that the overall conformation of the mutant receptors was similar to that of the native, although there were effects on the binding of certain antagonists. The effect of the mutations on agonist binding to the free receptor (uncoupled from G proteins) was determined in the presence of GTP (100 µM). This showed that there was no single mode of binding of catecholamine agonists to the receptor and that all three serine residues can participate in the binding of some agonists, possibly through hydrogen bonds to the catechol hydroxyl groups. Coupling of the mutant receptors to G proteins was assessed from agonist‐binding curves in the absence of GTP, when higher and lower affinity agonist‐binding sites were seen. Receptor/G protein coupling was generally unaffected by the Ala193 and Ala194 mutations, but the Ala197 mutation eliminated receptor/G protein coupling for some agonists. These data show that the interactions of agonists with the free and coupled forms of the receptor are different.

Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction

“…Mutation of serine 194 in the dopamine D2 receptor is reported to have little or no effect on spiperone binding. 18 The S194A mutated receptor, stably expressed in transfected C6 cells, was unable to inhibit hormone-stimulated elevation of cyclic AMP accumulation in response to dopamine stimulation. 18 While we find that mutation of serine 194 to alanine substantially reduces the signaling capacity of the dopamine D2 receptor, we still detect a modest ability of the D2R194A mutant receptor to modulate cAMP levels.…”

“…18 The S194A mutated receptor, stably expressed in transfected C6 cells, was unable to inhibit hormone-stimulated elevation of cyclic AMP accumulation in response to dopamine stimulation. 18 While we find that mutation of serine 194 to alanine substantially reduces the signaling capacity of the dopamine D2 receptor, we still detect a modest ability of the D2R194A mutant receptor to modulate cAMP levels. Since preliminary binding data suggested that there is not much difference in the ability of the two mutant D2R receptors to bind [ 3 H]spiperone, we have examined more fully the ability of the D2R80A mutant receptor to bind ligand and to function as a PET reporter gene in vivo.…”

“…Two mutations have been reported to uncouple ligand binding from subsequent D2R-mediated inhibition of cyclic AMP accumulation in cells. [16][17][18] Using standard site-directed mutagenesis procedures, we created cDNAs with the codon replacements D2R80A and D2R194A, shown in Figure 1. In D2R80A, codon 80 was changed from GAT (Asp) to GCT (Ala); in D2R194A, codon 194 was switched from TCC (Ser) to GCC (Ala).…”

“…The primary response evoked by dopamine binding to the dopamine D2 receptor is the activation of a Gi-protein complex that leads to inactivation of adenylyl cyclase and a consequent decrease in cellular cyclic AMP levels. Fortunately, two mutations have been described [16][17][18] that uncouple ligand binding of the dopamine D2 receptor to activation of the G␣i subunit of the Gi-protein complex signaling pathway that suppresses cyclic AMP synthesis in cells. We have investigated the utility of these two mutated D2R genes as PET reporter genes.…”

Role of Conserved Serine Residues in the Interaction of Agonists with D3 Dopamine Receptors

“…It has attracted much attention as a potential target for the design of novel antipsychotic drugs with reduced motor side effects (Sokoloff and Schwartz, 1995). The D 3 dopamine receptor is related structurally and functionally to the D 2 dopamine receptor, but a notable difference between the two is the markedly higher affinity of the D 3 receptor for certain agonists, e.g., dopamine, quinpirole, and 7-hydroxydipropylaminotetralin (7-OH-DPAT) (Neve and Neve, 1997).Agonist interaction with the D 2 dopamine receptor has been studied extensively using mutagenesis and modelling approaches, and this has identified three serine residues (Ser 193 , Ser 194 , and Ser 197 ) that are important for interaction of the catechol hydroxyl groups of dopamine and other catecholcontaining agonists with the receptor (Cox et al, 1992;Mansour et al, 1992;Woodward et al, 1996). The higher affinities of certain agonists for the D 3 receptor as compared with the D 2 receptor may be due to stronger interactions with the corresponding serine residues (Ser 192 , Ser 193 , and Ser 196 ), but the role of these serine residues has not been studied for the D 3 receptor.…”