Glycoprotein nature of D2 dopamine receptors.

The glycoprotein nature of the ligand binding subunit of photoaffinity-labeled striatal D2 receptors was investigated. Upon photolysis, [125I]N-azidophenethylspiperone covalently incorporated into a major band of Mr 94000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography following SDS-polyacrylamide gel electrophoresis. The exoglycosidase, neuraminidase, altered the electrophoretic mobility of the 94 kDa labeled band to 54 kDa with a slight modification in the binding affinity of [3H]spiperone. Endoglycosidase treatment (glycopeptidase-F) produced a further increase in the mobility of the 94 kDa peptide to approximately 43 kDa. A smaller specifically photolabeled D2 receptor peptide of 34 kDa does not contain terminal sialic acid but is an N-linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase-F to a peptide of approximately 23 kDa.

Cloning of two additional catecholamine receptors from rat brain.

An approach based on the polymerase chain reaction (PCR) was used to isolate additional members of the G-linked receptor family from a rat striatal lambda gtII cDNA library. Priming with one degenerate probe corresponding to highly conserved consensus sequences in the third transmembrane (TM) domain of 15 G-linked receptors and sequences in the phage vector resulted in one clone (G-13) encoding a dopamine D2 receptor variant with a 29 amino acid insert in the third cytoplasmic loop. In addition, the amino acid sequence encoded by clone G-36 contained conserved sequences characteristic of the G-linked class of receptors and displayed sequence homology in TM domains with the beta 2-adrenergic receptor (48%). Two conserved serine residues in TM5 postulated to be part of a ligand binding site in the adrenergic receptor, suggests that G-36 encodes a catecholaminergic receptor. Northern blot analysis confirmed the expression of G-36 in rat brain, but not in kidney, heart and lung. Several strong hybridizing bands to G-36 were obtained in both human and rat genomic DNA. The general PCR strategy employed here should prove to be extremely useful for the isolation of other members of the G-linked receptor family.

Novel AMPA receptor potentiators LY392098 and LY404187: effects on recombinant human AMPA receptors in vitro.

The present study describes the activity of two novel potent and selective AMPA receptor potentiator molecules LY392098 and LY404187. LY392098 and LY404187 enhance glutamate (100 microM) stimulated ion influx through recombinant homomeric human AMPA receptor ion channels, GluR1-4, with estimated EC(50) values of 1.77 microM (GluR1(i)), 0.22 microM (GluR2(i)), 0.56 microM (GluR2(o)), 1.89 microM (GluR3(i)) and 0.20 microM (GluR4(i)) for LY392098 and EC(50) values of 5.65 microM (GluR1(i)), 0.15 microM (GluR2(i)), 1.44 microM (GluR2(o)), 1.66 microM (GluR3(i)) and 0.21 microM (GluR4(i)) for LY404187. Neither compound affected ion influx in untransfected HEK293 cells or GluR transfected cells in the absence of glutamate. Both compounds were selective for activity at AMPA receptors, with no activity at human recombinant kainate receptors. Electrophysiological recordings demonstrated that glutamate (1 mM)-evoked inward currents in human GluR4 transfected HEK293 cells were potentiated by LY392098 and LY404187 at low concentrations (3-10 nM). In addition, both compounds removed glutamate-dependent desensitization of recombinant GluR4 AMPA receptors. These studies demonstrate that LY392098 and LY404187 allosterically potentiate responses mediated by human AMPA receptor ion channels expressed in HEK 293 cells in vitro.

Reactivity of normal and VMH-lesion rats to quinine-adulterated foods: negative evidence for negative finickiness.

Much of the evidence for the existence of a negative finickiness component in the ventromedial hypothalamic (VMH) syndrome comes from studies comparing the relative reactivities of VMH and normal rats to quinine adulteration. The present experiments addressed two major questions regarding the response of normal and VMH animals to quinine: (a) Do the anorexic properties of quinine depend on quinine's sensory properties (i.e., its bitter taste) or on its postingestive effects; (b) do VMH rats in fact overrespond to quinine adulteration? These issues were examined by comparing the feeding adjustments to quinine by VMH and normal animals in a sham-feeding situation and under normal feeding circumstances, on animals' initial exposure to this drug. The results were consistent with the view that the sensory properties of quinine alone were sufficient to induce large changes of food intake in both groups. In terms of whether lesion rats were more reactive to the taste of quinine, it is argued that previous research had measured reactivity in two dissimilar ways. The present data were used to illuminate how use of these two procedures for measuring reactivity lead to diametrically opposed conclusions regarding the existence of negative finickiness in the VMH syndrome. It is suggested that when the more appropriate measure of reactivity is adopted and when the confound of body weight differences between normal and VMH animals is eliminated, little evidence exists for a conclusion that VMH rats are more reactive than normals to quinine-adulterated foods.

Deglycosylation and proteolysis of photolabeled D2 dopamine receptors of the porcine anterior pituitary.

Dopamine D2 receptor binding subunits of the porcine anterior pituitary were visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The ligand binding subunit comprising the pituitary D2 dopamine receptor migrated as two distinct bands of apparent Mr approximately equal to 150,000 and 118,000, substantially higher than neuronal D2 receptor subunits from porcine or canine brain. The glycoprotein nature of pituitary D2 receptor binding subunits was investigated by the use of exo- and endo-glycosidase treatments and peptide mapping experiments. Photoaffinity labeled polypeptides of the anterior pituitary were susceptible to both neuraminidase and alpha-mannosidase digestion as indexed by their increased electrophoretic mobility on sodium dodecyl-sulfate polyacrylamide gels, and suggests the presence of both complex type and terminal mannose carbohydrate residues. Moreover, the additive effects of sequential treatment with these enzymes suggests that both types of carbohydrate chains are present on each receptor peptide. N-linked deglycosylation of pituitary D2 photolabeled receptors with glycopeptidase-F produced a further increase in the mobility of the labeled protein to apparent Mr approximately equal to 44,000, similar to that of deglycosylated D2 binding subunits of porcine and canine brain. Peptide mapping experiments following limited proteolysis with Staphylococcus aureus V8 proteinase and papain demonstrated that deglycosylated D2 dopamine receptors (Mr = 44,000), in different tissues and species, were homologous. Taken together, these data suggest that despite the differences in the overall molecular weight and tissue specific glycosylation pattern of pituitary D2 dopamine receptors, the primary structure of mammalian D2 receptors appears to be conserved.

Dopamine D2 receptors retain agonist high-affinity form and guanine nucleotide sensitivity after removal of sialic acid.

The ligand binding subunit of the D2 dopamine receptor (Mr approximately equal to 94,000) can be visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following removal of sialic acids with the exoglycosidase, neuraminidase, [125I]N-azidophenethylspiperone photoincorporated into a protein of Mr = 54,000 with the appropriate pharmacological profile for D2 receptors. The desialylated D2 receptor bound dopaminergic agonists with high affinity and was capable of coupling to a functional G-protein as indexed by: 1) pertussis-toxin mediated [32P]ADP ribosylation of proteins of Mr = 42,000 and 39,000, and 2) the conversion of the agonist high affinity form of D2 receptors to one displaying low affinity for agonists in the presence of guanine nucleotides. These data suggest that sialic acid residues do not contribute significantly to the ligand binding characteristics of D2 receptors despite the large change produced in the estimated molecular mass of the binding subunit.

Dopamine D-2 receptors in canine brain: ionic effects on [3H]neuroleptic binding.

In order to determine the effects of monovalent cations on the binding of 3H-neuroleptics to canine striatal dopamine D-2 receptors, a study of the effects of ions on the binding of two 3H-neuroleptics from different drug classes was undertaken. Dopamine D-2 receptors are selectively labeled in a sodium-sensitive manner by the benzamide ligand [3H]YM-09151-2. In the presence of 120 mM NaCl, [3H]YM-09151-2 had a dissociation constant of 55 pM and a maximal receptor density of 34 pmol/g of tissue. In the absence of NaCl, the dissociation constant was 440 pM with no change in the receptor density. The binding of [3H]YM-09151-2 (52 pM) was increased by 700% with 150 mM Na+, 440% with 500 mM Li+ and 290% with 500 mM K+. The ion concentrations producing half-maximal increases in binding were 4 mM Na+, 8.5 mM Li+ and 115 mM K+. The ionic strength control, N-methyl-D-glucamine, did not increase [3H]YM-09151-2 binding. [3H]Spiperone binding was much less affected by monovalent cations. Analysis of saturable binding showed that these changes were due to changes in binding affinity, independent of changes in receptor density. The sulfhydryl alkylating agent, N-ethylmaleimide, inactivated [3H]YM-09151-2 binding. Sodium ions at 120 mM protected approximately 40% of the susceptible sites while lithium and potassium ions (120 mM) did not. The anion exchange blocker, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS), has been shown to be capable of blocking the effect of Na+ on the binding of agonists to alpha 2-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

High affinity agonist binding to the dopamine D3 receptor: chimeric receptors delineate a role for intracellular domains.

The dopamine D3 receptor, although structurally similar to the dopamine D2 receptor, has 100-fold higher affinity for agonists such as dopamine and quinpirole, when these receptors are expressed in 293 cells. Additionally, the D3 receptor has generally lower affinity for several antagonists than does the D2 receptor. To determine which regions of the receptor account for these differences, chimeras between D2 and D3 receptors were constructed in which intracellular loops were exchanged between the two receptors. A D2 receptor containing the third intracellular loop (IL3) from the D3 receptor had 10-20-fold higher affinity for dopamine and quinpirole than did the wild-type D2 receptor. Conversely, the D3 receptor containing the IL3 of the D2 receptor had 15-30-fold lower affinity for agonists than did the wild-type D3 receptor. That is, in these chimeras the IL3 shifted agonist affinity in a direction consistent with the agonist affinity of the receptor from which the IL3 was derived. In contrast, antagonist binding was not significantly altered. Chimeras in which the second intracellular loop was switched between the D2 and D3 receptors had essentially unchanged affinity for both agonists and antagonists. The data presented here suggest that structural differences in the IL3 of the D2 and D3 receptors partially account for observed differences in agonist binding to these receptors.

Dopamine D1 receptors of the calf parathyroid gland: identification of a ligand binding subunit with lower apparent molecular weight but similar primary structure to neuronal D1-receptors.

The ligand binding subunit of the calf parathyroid D1 dopamine receptor was visualized by autoradiography following photoaffinity labeling with (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4'-azidophenyl)-2,3,4,5- tetrahydro-1H-benzazepine ([125I]IMAB) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein comprising the D1 binding subunit migrated with an apparent Mr approximately equal to 62,000. Photoincorporation of [125I]IMAB into the Mr approximately equal to 62,000 polypeptide required the presence of protease inhibitors and was stereoselectively antagonized by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The electrophoretic mobility of the [125I]IMAB-labeled receptor was not altered by the absence or presence of urea or thiol-reducing/oxidizing reagents. The Mr approximately equal to 62,000 protein representing the ligand binding subunit of bovine parathyroid D1 receptors corresponds to one of three D1 receptor binding subunits (Mr = 74,000, 62,000, and 51,000) identified in bovine brain. Peptide map comparisons of radiolabeled D1 receptors from calf parathyroid and brain following limited proteolytic digestion with Staphylococcus aureus V8 and papain revealed marked structural similarities. These data suggest that, despite tissue-specific differences in overall molecular weight, both parathyroid and neuronal D1 dopamine binding subunits appear to be pharmacologically and structurally homologous and that the molecular mechanism(s) responsible for the apparent lack of a one to one correspondence in the subunit composition of the D1 receptor in these tissues probably reflect(s) tissue-specific posttranslational modifications.

Dopamine D2 receptor binding subunits of Mr congruent to 140,000 and 94,000 in brain: deglycosylation yields a common unit of Mr congruent to 44,000.

The ligand-binding subunit of the porcine striatal dopamine D2 receptor was identified by photoaffinity labeling with [125I]N-azidophenethylspiperone ([125I]NAPS). Upon photolysis, [125I]NAPS covalently incorporated into a broad band of apparent Mr congruent 140,000 with an appropriate pharmacological profile for D2 receptors as assessed by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Smaller subunits of apparent Mr congruent 94,000 and 34,000 were specifically labeled by [125I]NAPS with an appropriate D2 receptor profile and were similar to the major ligand-binding subunits of photoaffinity-labeled canine striatal D2 receptors. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of the Mr congruent to 140,000/94,000 subunits upon denaturing electrophoresis in either the absence or presence of thiol-reducing/alkylating reagents. In order to investigate the possible basis for the existence of these high molecular weight forms of the D2 receptor, we assessed the carbohydrate nature of photolabeled D2 ligand-binding subunits by the use of lectin affinity chromatography and specific exo- and endoglycosidase treatments. Both photoaffinity-labeled D2 receptor proteins from porcine striatum (Mr congruent to 140,000 and 94,000) were glycoproteins as indexed by their absorption and specific elution from wheat germ agglutinin lectin resins. The exoglycosidase neuraminidase altered the electrophoretic mobility of both the Mr congruent to 140,000 and 94,000 labeled subunits to a single band of apparent Mr congruent to 51,000. Prior removal of sialic acid residues did not alter the reversible binding characteristics of [3H]spiperone to D2 receptors. Complete removal of receptor-associated N-linked carbohydrate by the endoglycosidase glycopeptidase F (peptide-N4[N-acetyl-beta-glucosaminyl]asparagine amidase) produced a further increase in the mobility of the Mr congruent to 51,000 subunit to apparent Mr congruent to 44,000. The porcine Mr congruent to 34,000 photolabeled peptide is an N-linked glycoprotein as assessed by lectin affinity chromatography and susceptibility to digestion by glycopeptidase F to a peptide of apparent Mr congruent to 23,000.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycoprotein nature of dopamine D1 receptors in the brain and parathyroid gland.

Dopamine D1 receptors can be covalently labeled with the photo-affinity ligand (+-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetrah yd ro-1H-3-benzazepine ([125I]IMAB) and visualized following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. In brain membranes, [125I]IMAB labels a polypeptide of apparent Mr approximately equal to 74,000 as the major ligand binding subunit of D1 receptors and two minor polypeptides of Mr approximately equal to 64,000 and 52,000. In contrast, [125I]IMAB labels a single polypeptide of apparent Mr approximately equal to 64,000 in bovine parathyroid glands. In this study, the carbohydrate nature of dopamine D1 receptors from the brain and parathyroid gland were examined using specific exo- and endoglycosidases and lectin affinity chromatography. [125I]IMAB-labeled brain and parathyroid D1 receptors were sensitive to treatment with the exoglycosidases neuraminidase or alpha-mannosidase, suggestive of the existence of terminal sialic acid and oligomannose residues. Photolabeled D1 receptor polypeptides are not however, associated with distinct populations of complex-type or high mannose-containing carbohydrate chains because 1) wheat germ agglutinin and concanavalin A lectin chromatography of solubilized and photolabeled neuronal D1 receptors followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed no differences in the electrophoretic mobility of column pass-through and specifically eluted [125I]IMAB-labeled polypeptides, and 2) [125I]IMAB-labeled D1 receptors specifically bound to and eluted from concanavalin A-Sepharose were neuraminidase sensitive, indicative of the colocalization of oligomannose- and complex-type glycans. Removal of these terminal glycan residues did not affect the binding of [3H]SCH 23390 to dopamine D1 receptors. Complete N-linked deglycosylation of photolabeled D1 receptors from both the brain and parathyroid with peptide N-glycosidase F resulted in the migration of a single major labeled polypeptide of apparent Mr approximately equal to 46,000. These data suggest that, despite differences observed in the electrophoretic mobility and glycosylation patterns of brain and parathyroid D1 receptor polypeptides, the protein backbones of central and peripheral dopamine D1 receptors display similar if not identical molecular weights.

Silencing of the constitutive activity of the dopamine D1B receptor. Reciprocal mutations between D1 receptor subtypes delineate residues underlying activation properties.

Recently, we have shown that the dopamine D1B/D5 receptor displays binding and coupling properties that are reminiscent of those of the constitutively activated G protein-coupled receptors when compared with the related D1A/D1 receptor subtype (Tiberi, M., and Caron, M. G. (1994) J. Biol. Chem. 269, 27925-27931). The carboxyl-terminal region of the third cytoplasmic loop of several G protein-coupled receptors has been demonstrated to be important for the regulation of the equilibrium between inactive and active receptor conformations. In this cytoplasmic region, the primary structure of dopamine D1A and D1B receptors differs by only two residues: Phe264/Arg266 are present in D1A receptor compared with Ile288/Lys290 in the D1B receptor. To investigate whether these structural differences could account for the distinct binding and coupling properties of these dopamine receptor subtypes, we swapped the variant residues located in the carboxyl-terminal region by site-directed mutagenesis. The exchange of the D1A receptor residue Phe264 by the D1B receptor counterpart isoleucine led to a D1A receptor mutant exhibiting D1B-like constitutive properties. In contrast, substitution of D1B receptor Ile288 by the D1A receptor counterpart phenylalanine resulted in a loss of constitutive activation of the D1B receptor with binding and coupling properties similar to the D1A receptor. The Arg/Lys substitution had no effect on the function of either receptor. These results demonstrate that the carboxyl-terminal region, and in particular residue Ile288, is a major determinant of the constitutive activity of the dopamine D1B receptor. Moreover, these results establish that not only can agonist-independent activity of a receptor be induced, but when given the appropriate mutation, it can be reversed or silenced.

Molecular cloning, stable expression and desensitization of the human dopamine D1b/D5 receptor.

The sub-family of dopamine D1-like receptors is now known to be comprised of at least two members: the originally cloned D1 receptor (herein referred to as the D1a receptor) and a related receptor referred to as the D1b, D1 beta or D5 dopamine receptor (herein referred to as the D1b/D5 receptor). Here, we characterize the D1b/D5 receptor expressed transiently in COS-7 cells and permanently in Ltk- cells. Transiently expressed human D1b/D5 receptors bind the D1 specific ligand [125I]SCH 23982 saturably and with high affinity (KD = 500 pM). Competition for [125I]SCH 23982 binding to rat D1b/D5 and human D1a and D1b/D5 receptors supports the contention that the two D1b/D5 receptors are species homologues. Furthermore, in COS-7 cells, as previously observed, dopamine competes for the binding of [125I]SCH 23982 to human D1b/D5 receptors with a higher affinity than that seen at the human D1a receptor. These results are similar to those seen in Ltk- cells permanently transfected with the human D1b/D5 receptor. In these cells, dopamine competition for [125I]SCH 23982 binding is complex, sensitive to guanine nucleotides and of a higher affinity than that observed for dopamine binding to the human D1a receptor expressed in these same cells. In both D1a and D1b/D5 expressing Ltk- cells, dopamine stimulates adenylyl cyclase with an EC50 of approximately 200 nM. Furthermore, preincubation of Ltk- cells expressing the D1a and D1b/D5 receptors with dopamine results in desensitization of the response of adenylyl cyclase to subsequent agonist stimulation.

Photoaffinity labeling of dopamine D1 receptors.

A high-affinity radioiodinated D1 receptor photoaffinity probe, (+/-)-7-[125I]iodo-8-hydroxy-3-methyl-1-(4-azidophenyl)-2,3,4,5-tetra hyd ro- 1H-3-benzazepine ([125I]IMAB), has been synthesized and characterized. In the absence of light, [125I]IMAB bound in a saturable and reversible manner to sites in canine brain striatal membranes with high affinity (KD approximately equal to 220 pM). The binding of [125I]IMAB was stereoselectively and competitively inhibited by dopaminergic agonists and antagonists with an appropriate pharmacological specificity for D1 receptors. The ligand binding subunit of the dopamine D1 receptor was visualized by autoradiography following photoaffinity labeling with [125I]IMAB and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon photolysis, [125I]IMAB incorporated into a protein of apparent agents in a stereoselective manner with a potency order typical of dopamine D1 receptors. In addition, smaller subunits of apparent Mr 62,000 and 51,000 were also specifically labeled by [125I]IMAB in these species. Photoaffinity labeling in the absence or presence of multiple protease inhibitors did not alter the migration pattern of [125I]IMAB-labeled subunits upon denaturing electrophoresis in both the absence or presence of urea or thiol reducing/oxidizing reagents. [125I]IMAB should prove to be a useful tool for the subsequent molecular characterization of the D1 receptor from various sources and under differing pathophysiological states.

Cloning, molecular characterization, and chromosomal assignment of a gene encoding a second D1 dopamine receptor subtype: differential expression pattern in rat brain compared with the D1A receptor.

Multiple D1 dopaminergic receptor subtypes have been postulated on the basis of pharmacological, biochemical, and genetic studies. We describe the isolation and characterization of a rat gene encoding a dopamine receptor that is structurally and functionally similar to the D1 dopamine receptor. The coding region, which is intronless, encodes a protein of 475 amino acids (Mr 52,834) with structural features that are consistent with receptors coupled to guanine nucleotide-binding regulatory proteins. The expressed protein binds dopaminergic ligands and mediates stimulation of adenylyl cyclase with pharmacological properties similar to those of the D1 dopamine receptor. The gene encoding the human homologue of this receptor subtype is located to the short arm of chromosome 4 (4p16.3), the same region as the Huntington disease gene. In striking contrast to the previously cloned D1 receptor, little or no mRNA for the receptor described here was observed in striatum, nucleus accumbens, olfactory tubercle, and frontal cortex. High levels of mRNA for this receptor were found in distinct layers of the hippocampus, the mammillary nuclei, and the anterior pretectal nuclei, brain regions that have been shown to exhibit little or no D1 dopamine receptor binding. On the basis of its properties we propose that this dopamine receptor subtype be called D1B.