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.

Three conserved serine residues (Ser193, Ser194, and Ser197) in transmembrane spanning region (TM) V of the D2 dopamine receptor have been mutated to alanine, individually and in combination, to explore their role in ligand binding and G protein coupling. The multiple Ser -->Ala mutations had no effect on the binding of most antagonists tested, including [3H]spiperone, suggesting that the multiple mutations did not affect the overall conformation of the receptor protein. Double or triple mutants containing an Ala197 mutation showed a decrease in affinity for domperidone, whereas Ala193 mutants showed an increased affinity for a substituted benzamide, remoxipride. However, dopamine showed large decreases in affinity (>20-fold) for each multiple mutant receptor containing the Ser193Ala mutation, and the high-affinity (coupled) state of the receptor (in the absence of GTP) could not be detected for any of the multiple mutants. A series of monohydroxylated phenylethylamines and aminotetralins was tested for their binding to the native and multiple mutant D2 dopamine receptors. The results obtained suggest that Ser193 interacts with the hydroxyl of S-5-hydroxy-2-dipropylaminotetralin (OH-DPAT) and Ser197 with the hydroxyl of R-5-OH-DPAT. We predict that Ser193 interacts with the hydroxyl of R-7-OH-DPAT and the 3-hydroxyl (m-hydroxyl) of dopamine. Therefore, the conserved serine residues in TMV of the D2 dopamine receptor are involved in hydrogen bonding interactions with selected antagonists and most agonists tested and also enable agonists to stabilise receptor-G protein coupling.

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser(51)Ala site-directed mutant of eIF2alpha, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2alpha by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single- and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2alpha Ser(51)Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2alpha protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2alpha phosphorylation in cells transfected with the mutant eIF2alpha construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser(51)Ala or wild-type eIF2alpha proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.

The dopamine receptors belong to the superfamily of G-protein-linked receptors, which couple changes in cellular metabolism in response to neurotransmitters, peptide hormones and other factors to intracellular effector enzymes by interaction with a guanine-nucleotide-binding protein. G-protein-linked receptors are composed of a single polypeptide chain with seven relatively hydrophobic domains presumed to form membrane-spanning a-helices, interspersed by three sets of intra-and extra-cellular loops. The ligand-binding site is thought to be formed by the bundling of these seven a-helices [ 11, based on the physical studies of the evolutionarily-related proteins bacteriorhodopsin [2] and rhodopsin [3] and the results of mutational analysis of amino acid residues contained within these putative a-helical domains (see, for example, [4]). The high conservation of amino acids embedded in the membrane within this superfamily of receptors has suggested that these amino acids play a key functional or structural role in the action of this class of receptors. By understanding the general principles underlying common structurefunction relationships within this superfamily of receptors, it is hoped that a unifying hypothesis may be developed of how ligands are recognized by these receptors and the mechanism by which G-proteins are activated and elicit a secondmessenger response.Receptors for the neurotransmitter dopamine fall into two subgroups on the basis of biochemical, pharmacological and physiological studies, and are derived from two divergent gene families (reviewed Abbreviations used: 5,6-ADTN, ( * )-2-amino-5,6-dihydroxy-1,2,3,4-tetrahydronaphthalene; 6,7-ADTN, ( k )-Z-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene; SB, substituted benzamide; TM, transmembrane. 'To whom correspondence should be addressed.in [S]). The D,-like receptor subfamily contains the D, and D, isoforms, which are encoded by genes which lack introns, and have short third intracellular loops and long C-terminal tails. The D,-like receptor genes (D,, D, and D4) contain introns, and have long third intracellular loops and short C-terminal tails. These receptor domains have been implicated in G-protein coupling and may account (in part) for receptors of the same subfamily interacting with a subset of G-proteins to elicit a similar functional response [S]. The neurotransmitter dopamine has been implicated in the aetiology of brain disorders such as Parkinson's disease and schizophrenia, and dopamine receptors provide important targets for anti-parkinsonian and anti-psychotic drugs (reviewed in [6]). Elucidation of the molecular mechanisms underlying drug-receptor interaction and receptor function or response wil facilitate the design and development of new therapeutic agents which may act at these receptors. The availability of cloned receptors has allowed mutational analysis of these receptors to be performed, which has been important for determining the molecular basis of dopamine-receptor function. Using site-directed mutagenesis, the role of i...

The D, dopamine receptor is a member of the large family of G-protein linked receptors, consisting of 7 hydrophobic domains presumed to form transmembrane (TM) domains linked by intia-and extra-cellular loops (1). In common with other members of this receptor superfamily, particularly those which bind cationic amines, several conserved amino acids residues have been identified in TM3 (aspartic acid) and TM5 (serines) which may be important for the specificity with which different agonist drugs bind these receptors (2,3). Similar studies in the rat D, dopamine receptor have shown that these residues may also affect the binding of antagonist drugs (4,5).In this study, three conserved serine residues in TM5 of the dopamine receptor (Ser 193, 194 and 197) have been mutated to alanines using site-directed mutagenesis, and expressed transiently in COS-7 cells. Membranes prepared from transfected cells were then used for saturation binding analysis using the radioligand ['HI-spiperone (0.08 nM -2 nM), and specific binding was determined by (t ) butaclamol. The effects of the Ala mutations on the binding of a range of antagonist ligands representing both the classical and substituted benzamide class of drugs were also analysed by performing competition assays in the presence of 0.25 nM ['HI-spiperone. The reaction volume was made up to Iml in buffer (20 mM HEPES pH7.4, 1mM EDTA, ImM EGTA and 120mM NaCI), and incubated for 45 minutes at 25°C. The membranes were harvested onto Whatman GF/C glass fibre filters using a Brandel cell harvester, and the bound activity determined by liquid scintillation counting.