Detergent-free extraction of a functional low-expressing GPCR from a human cell line.

Dopamine receptors (DRs) are class A G-Protein Coupled Receptors (GPCRs) prevalent in the central nervous system (CNS). These receptors mediate physiological functions ranging from voluntary movement and reward recognition to hormonal regulation and hypertension. Drugs targeting dopaminergic neurotransmission have been employed to treat several neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, Huntington's disease, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome. In vivo, incorporation of GPCRs into lipid membranes is known to be key to their biological function and, by inference, maintenance of their tertiary structure. A further significant challenge in the structural and biochemical characterization of human DRs is their low levels of expression in mammalian cells. Thus, the purification and enrichment of DRs whilst retaining their structural integrity and function is highly desirable for biophysical studies. A promising new approach is the use of styrene-maleic acid (SMA) copolymer to solubilize GPCRs directly in their native environment, to produce polymer-assembled Lipodisqs (LQs). We have developed a novel methodology to yield detergent-free D1-containing Lipodisqs directly from HEK293f cells expressing wild-type human dopamine receptor 1 (D1). We demonstrate that D1 in the Lipodisq retains activity comparable to that in the native environment and report, for the first time, the affinity constant for the interaction of the peptide neurotransmitter neurotensin (NT) with D1, in the native state.

Biotin ergopeptide probes for dopamine receptors.

The incorporation of chemical modifications into the structure of bioactive compounds is often difficult because the biological properties of the new molecules must be retained with respect to the native ligand. Ergopeptides, with their high affinities at D(1) and D(2) dopamine receptors, are particularly complex examples. Here, we report the systematic derivatization of two ergopeptides with different peptide-based spacers and their evaluation by radioligand binding assays. Selected spacer-containing ergopeptides with minimal biological alteration and a proper anchoring point were further derivatized with a biotin reporter. Detailed characterization studies identified 13 as a biotin ergopeptide maintaining high affinity and agonist behavior at dopamine receptors, being a useful tool for the study of heteromers involving D(1)R, D(2)R, or D(3)R.

Higher basal serine phosphorylation of D1A receptors in proximal tubules of old Fischer 344 rats.

Dopamine (DA) and D1-like receptor agonists promote an increase in Na excretion by means of activation of the D1-like receptor signaling cascade and subsequent inhibition of the Na/H exchanger and Na-K-ATPase in renal proximal tubules. Recently, our laboratory reported that DA and the D1-like receptor agonist failed to inhibit Na-K-ATPase activity in old Fischer 344 rats because of uncoupling of D1A receptors from G proteins and that this abnormality led to a diminished natriuretic response to DA in old Fischer 344 rats. In this study, we have tested the hypothesis that the mechanism of this uncoupling may be an altered phosphorylation of D1A receptors in old rats. In experiments performed in renal cortical slices, both DA and SKF-38393, a D1-like receptor agonist, increased the serine phosphorylation of D1A receptors in adult (6 mo) but not old (24 mo) rats. Interestingly, the basal serine phosphorylation of D1A receptors was higher in old than in adult rats. Competition ligand binding ([3H]SCH-23390) experiments on the D1-like receptor in adult and old rats with fenoldopam, a D1-like receptor agonist, revealed the presence of two affinity states of the receptors. There was a rightward shift in the agonist displacement of the ligand in old compared with adult rats, as reflected in the IC50 values (adult vs. old, 7.46 x 10(-9) +/- 2.26 vs. 7.93 x 10(-7) +/- 1.33 M). Also, there was a reduction in agonist affinity in the low-affinity receptors in old compared with adult rats (IC50, adult vs. old, 5.67 x 10(-5) +/- 1.33 vs. 12.60 x 10(-5) +/- 6.50 M). Moreover, the abundance of D1A receptor proteins was approximately 47% lower in the membranes of old compared with adult rats. We speculate that higher basal serine phosphorylation of D1A receptors may have rendered the D1A receptor uncoupled from G protein, leading to a reduced agonist affinity state and thus diminished natriuretic response to DA in old rats.

Putative leech dopamine1-like receptor molecular characterization: sequence homologies between dopamine and serotonin leech CNS receptors explain pharmacological cross-reactivities.

The biochemical characterization of a serotonin (5HT) receptor and the cloning of a dopamine (DA) receptor in the central nervous system (CNS) of the leech, Theromyzon tessulatum, is presented. Additionally, DA and 5HT binding sites were examined in the CNS by Scatchard analysis which showed a single, relatively high-affinity binding site with a Kd 1.1 nM and a Bmax 126+18 fmol/mg protein for [3H]DA and a Kd 2.1 nM and a Bmax 225 fmol/mg protein for [3H]5HT. The first 88 amino acids of the 5HT receptor, isolated by a 5HT-affinity column followed by anion exchange chromatography and C3 reverse-phase HPLC exhibited a 43% sequence homology with Lymnaea stagnalis 5HT-receptor. The isolated DA receptor revealed a single protein of 45 kDa with an anti-D1-R in Western blot. The first 80 N-terminal amino acid residues and a trypsin digested fragment of 31 residues were obtained, and based on these sequencing data, a molecular biology strategy using reverse transcriptase-polymerase chain reaction, was developed. An amplified 1-kb segment was obtained. The complete deduced sequence of 416 amino acid residues exhibited about 30.6% sequence homology with the vertebrate D1 receptor family. Moreover, we further demonstrate that the leech 5HT and DA receptors also exhibit 30% sequence identity with each other, explaining their pharmacological cross-reactivity. Finally, anti-D1-R immunocytochemistry revealed positive structures in the peripheral and central nervous system, e.g., neurons, sensory fibers and immune cells. This is the first biochemical and molecular characterization of a DA receptor in leeches.

The human D1A dopamine receptor: heterologous expression in Saccharomyces cerevisiae and purification of the functional receptor.

Functional human D1A dopamine receptor has been expressed in Saccharomyces cerevisiae. The primary sequence of the receptor was modified to include two affinity tags at the C-terminus of the protein, a FLAG tag (DYKDDDDK), and a His6 tag (HHHHHH). These modifications allowed for purification to near homogeneity using immobilized metal affinity chromatography and immunoaffinity chromatography. Radioligand binding demonstrated that the purified and reconstituted receptor binds the antagonist [3H]SCH23390 with an affinity (KD = 8.0 +/- 3.2 nM) comparable to that of the native receptor.

Transient expression of an atypical D1-like dopamine receptor system during avian retina differentiation

Intact cultured retina cells from chick embryos at stage E9C5 (cultures initiated with retinae from 9-day old embryos followed by 5 days in culture), preincubated with 2 nM unlabelled SCH 23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) for 20 to 60 min at 37 degrees Celsius and then washed 5 to 25 times (approximately 1.5 min/wash) with 2 ml SCH 23390 free medium, responded to dopamine with cAMP accumulation that corresponded to 30-50 per cent of the dopamine-promoted cAMP accumulation observed in untreated cells or in cells exposed to the inactive isomer of SCH 23390. Therefore, 50 to 70 per cent of the dopamine response of SCH 23390-pretreated cells was inhibited after extensive washings of the cultures. At E9C12 the fraction of the dopamine response that remained inhibited by SCH 23390 after the washings declined to 30 per cent of the control cultures or the cultures exposed to the SCH 23390 enantiomer. Cultures at stage E9C5 treated with SCH 23390 followed by extensive washings as above and then used for measuring the number of [3H]-SCH 23390 specific binding sites revealed that 60 per cent of the sites did not interact with the tritiated compound when compared to untreated cultures or to cultures preincubated with the inactive isomer of SCH 23390. When E9C12 cultures were subjected to the same experimental protocol less than 10 per cent of D1-like sites did not interact with [3H]-SCH 23390 after the cells had been exposed to the unlabelled compound. Dissociated cells prepared from intact retinae obtained from 12-13-day old embryos also displayed a subpopulation of D1-like sites that interacted irreversibly with SCH 23390 in a stereospecific way. These sites corresponded to 25 per cent of the total number of D1-like sites present in the retina at this developmental stage. In retina cells obtained from one-day old posthatched chicks these sites were no longer detected. These data show that cultured retina cells as well as cells obtained from retina developing in ovo display two populations of D1-like receptors. One interacts irreversibly with SCH 23390 and is present only in the undifferentiated tissue or in cells at the early stages of culture and the other has a lower affinity for SCH 23390 with which its interaction follows reversible kinetics. These sites are present throughout the differentiation stages studied.

Differences in photoaffinity labeling of DA1 receptors in renal proximal tubules from normotensive rat and SHR.

Renal DA1 dopamine receptors in proximal tubule membranes of normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) were characterized with the novel D1 dopamine receptor-selective photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5- tetrahydro-1H-3-benzazepine ([125I]MAB). Under nonphotolyzing conditions, saturation studies showed that [125I]MAB bound with similar affinity to DA1 dopamine receptors in both WKY [dissociation constant (Kd) = 16.3 nM] and SHR (Kd = 19.5 nM). At photolysis, [125I]MAB was irreversibly incorporated into a single major protein of 74,000 Da in both WKY and SHR. DA1-selective antagonists blocked photolabeling of DA1 sites with similar efficiency and specificity in SHR and WKY. However, under identical assay conditions, dopaminergic agonists were unable to block photoincorporation of [125I]MAB in SHR but not in WKY. This pattern of labeling of DA1 sites by [125I]MAB may suggest the presence of defective agonist, but not antagonist, binding domains on the receptor in SHR but not in WKY rats.

Induction of G protein-independent agonist high-affinity binding sites of D-1 dopamine receptors by beta-mercaptoethanol.

We have purified the D-1 dopamine receptor 8200-fold to 78% purity from rat striatal membranes. Critical to this purification was the N-ethylmaleimide (NEM)-mediated alkylation of all endogenous sulfhydryl groups, except those associated with the D-1 dopamine receptors, which were protected by the D-1 agonist SKF R-38393. Such NEM treatment of D-1 receptors abolished all agonist high-affinity binding sites of the receptors, but did not alter the antagonist binding properties. When NEM-treated D-1 receptors were affinity-purified by mercury-agarose columns, the pharmacological properties of these purified receptors were examined, after removal of beta-mercaptoethanol (beta ME), which was used for elution of receptors from the affinity column. Purified D-1 receptors displayed typical dopaminergic antagonist binding values; however, agonists bound to the purified receptors with only high-affinity binding values, despite the prior absence of high-affinity sites in crude soluble extracts of NEM-treated receptors. The agonist high-affinity binding of purified D-1 receptors was insensitive to modulation by the GTP analog Gpp(NH)p and occurred in the absence of any G proteins. These Gpp(NH)p-insensitive high-affinity sites appeared to be induced by beta ME, since similar high-affinity binding was also restored by beta ME to crude soluble and membrane-bound receptors, which had been pretreated with NEM. The ability of D-1 dopamine receptors to bind with high-affinity to agonists in the absence of functionally active G proteins may be an intrinsic property of the reduced state of D-1 dopamine receptors.

Site of dopamine D1 receptor binding to Gs protein mapped with synthetic peptides.

We have synthesized peptides corresponding to the known sequence of the rat dopamine D1 receptor and measured the effects on binding of the Gs protein to the activated receptor. Peptides corresponding to the second and third cytoplasmic loop (amino acids 160-180 and 260-276), as well as the N-terminal part of the carboxy terminus (amino acids 375-391) were effective in preventing Gs binding to the activated receptor. Peptides representing other surface regions, e.g., the first cytoplasmic loop (87-100), the C-terminus (400-420 and 440-460) and all extracellular surface peptides (6-22, 120-137, 210-224 and 338-352) are not involved in rat dopamine D1 receptor-Gs protein interaction.

Specific phospholipid requirements for the solubilization and reconstitution of D-1 dopamine receptors from striatal membranes.

We have reported the solubilization and reconstitution of functional D-1 dopamine receptors from rat striatal tissue, using sodium cholate as detergent [Sidhu, A. (1988) Biochemistry 27, 8768-8776]. Critical to our method of extraction was the absolute requirement for the persistent presence of a crude extract of phospholipids (PLs) from bovine brain, during both solubilization of membranes and reconstitution of the soluble extract into PL vesicles. In the absence of PLs, fewer than 10% of the receptors were recovered, while in the presence of PLs, 40% of the receptors were reconstituted into vesicles. To probe the composition of PLs required by D-1 dopamine receptors during these extraction procedures, specific PLs of a defined composition were used during either solubilization or reconstitution alone or during both solubilization and reconstitution. Phosphatidylcholine (PC), when used during the solubilization procedure alone or during both solubilization and reconstitution, resulted in recovery of 41-48% of the D-1 dopamine receptors but was 3.7-fold less effective when present during reconstitution alone (11%). Phosphatidylethanolamine (PE), when used during reconstitution alone, resulted in recovery of nearly 25% of the D-1 dopamine receptors. When PE was present during either solubilization or both solubilization and reconstitution, 6-11% of the receptors were recovered. If PE was used with PC in ratios of 1:1 or 2:1, respectively, 28-38% of the receptors were recovered. When PL vesicles of PE:PC were present in ratios of 1:2 during both solubilization and reconstitution, the maximum theoretical (74-87%) recovery of total receptor binding sites was achieved.(ABSTRACT TRUNCATED AT 250 WORDS)