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Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp(2.50) and Asn(7.49) residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn(1.50(53)) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.

The behavioral effects of augmenting dopamine D1 receptor expression in the brain were investigated in mice incorporating additional copies of the mouse D1 receptor gene. Two transgenic lines showed increases in brain D1 receptor binding sites, which were greatest in extrastriatal regions. The full D1 agonist SKF 81297, when administered systemically to control animals, stimulated a dose-dependent increase in locomotor activity. In contrast, in D1 receptor overexpressing transgenic mice, this drug caused a marked suppression of locomotion due to a decrease in the frequency of movement initiation. Amphetamine and cocaine induced comparable locomotor activation in both transgenic animals and their control littermates. In the transgenic animals, D1 agonist-induced rearing and climbing behaviors were suppressed. However, on rotarod testing, the agonist-treated transgenic and control mice performed comparably, indicating that sensorimotor coordination was unaffected. These studies demonstrate that altering the levels of D1 receptor expression reverses the effects of D1 agonism on locomotor initiation and rearing., (Copyright 1999 Academic Press.)

Increased expression and nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are early, critical events in several forms of apoptosis. In order to investigate the subcellular trafficking of GAPDH in vivo, the localization of a GAPDH-green fluorescent protein (GFP) fusion was studied in PC12, HEK 293 and COS-1 cells. In control cells, fusion protein autofluorescence was largely restricted to the cytoplasm, rather than the nuclear concentration favored by GFP alone. In contrast, as early as 30 min after an insult, nuclear fluorescence increased in all cell lines studied. The fusion protein redistribution paralleled the dynamics of endogenous GAPDH. These data suggest that some nuclear GAPDH observed during apoptosis represents protein previously resident in the cytosol. This construct provides an in vivo monitor for an early change in apoptosis.

An Arg present in the third transmembrane domain of all rhodopsin-like G-protein-coupled receptors is required for efficient signal transduction. Mutation of this Arg in the gonadotropin-releasing hormone receptor to Gln, His, or Lys abolished or severely impaired agonist-stimulated inositol phosphate generation, consistent with Arg having a role in receptor activation. To investigate the contribution of the surrounding structural domain in the actions of the conserved Arg, an integrated microdomain modeling and mutagenesis approach has been utilized. Two conserved residues that constrain the Arg side chain to a limited number of conformations have been identified. In the inactive wild-type receptor, the Arg side chain is proposed to form an ionic interaction with Asp3.49(138). Experimental results for the Asp3. 49(138) --> Asn mutant receptor show a modestly enhanced receptor efficiency, consistent with the hypothesis that weakening the Asp3. 49(138)-Arg3.50(139) interaction by protonation of the Asp or by the mutation to Asn favors activation. With activation, the Asp3. 49(138)-Arg3.50(139) ionic bond would break, and the unrestrained Arg would be prevented from orienting itself toward the water phase by a steric clash with Ile3.54(143). The mutation Ile3.54(143) --> Ala, which eliminates this clash in simulations, causes a marked reduction in measured receptor signaling efficiency, implying that solvation of Arg3.50(139) prevents it from functioning in the activation of the receptor. These data are consistent with residues Asp3.49(138) and Ile3.54(143) forming a structural motif, which helps position Arg in its appropriate inactive and active receptor conformations.

G-protein-coupled receptors of the rhodopsin family transduce many important neural and endocrine signals. These receptors activate heterotrimeric G proteins and in many cases also cause activation of phospholipase D, an enzyme that can be controlled by the small G proteins ARF and RhoA. Here we show that the activation of phospholipase D that is induced by many, but not all, Ca2+-mobilizing G-protein-coupled receptors is sensitive to inhibitors of ARF and of RhoA. Receptors of this type were co-immunoprecipitated with ARF or RhoA on exposure to agonists, and the effects of GTP analogues on ligand binding to the receptor changed to a profile that is characteristic of small G proteins. These receptors contain the amino-acid sequence AsnProXXTyr in their seventh transmembrane domain, whereas receptors capable of activating phospholipase D without involving ARF contain the sequence AspProXXTyr. Mutation of this latter sequence to AsnProXXTyr in the gonadotropin-releasing hormone receptor conferred sensitivity to an inhibitor of ARF, and the reciprocal mutation in the 5-HT2A receptor for 5-hydroxy-tryptamine reduced its sensitivity to the inhibitor. Receptors carrying the AsnProXXTyr motif thus seem to form functional complexes with ARF and RhoA.

We have characterized pharmacological properties of inositol trisphosphate (InsP3)-mediated calcium entry pathway in Xenopus oocytes via activation of Ca(2+)-dependent Cl- channels (ICl, Ca) as a sensitive indicator for increase in cytosolic [Ca2+]. This type of Ca2+ entry mechanism is known as a capacitative Ca2+ entry (CCE). Voltage-clamped oocytes were maintained in Ca(2+)-free medium and injected with InsP3 which depleted the InsP3-sensitive Ca2+ stores. 10-20 min later, the oocytes were exposed, at 2-3 min intervals, to 5 mM Ca(2+)-containing medium for 5-10 s which evoked repeated inward Cl- current. No effect of external Ca2+ was apparent before InsP3 injection. To determine the pharmacological characteristics of CCE, oocytes were incubated with various chemical agents in Ca(2+)-free solution and exposed to Ca2+ again in presence of the chemical. It was found that organic Ca2+ channel blockers were relatively ineffective in blocking CCE while the inorganic Ca2+ channel blocker La3+ was most efficient in blocking the current. Attempts to measure conductance increase when the Cl- channels were blocked during activation of Ca2+ influx were unsuccessful. Therefore we tested the hypothesis that the Ca2+ influx is mediated via a Ca-H transporter. Lowering the external pH (to pH 6.5) or application of the protonophore carbonylcyanide p-trifluoromethoxyphenyl hydrazone (EC50 = 2 x 10(-8) M) effectively blocked CCE. Since Ca-H countertransport in the plasma membrane is coupled to Ca2+ extrusion by Ca-ATPase in vascular smooth muscle we suggest that the capacitative Ca2+ entry in Xenopus oocytes may possibly arise from slippage of plasma membrane Ca-ATPase coupled to proton countertransport, a mechanism reported in a variety of cells. Ca2+ slippage may arise from the large Ca2+ gradient produced by the Ca2+ depletion protocol.

An important determinant of the neurobehavioral responses induced by a drug is its relative receptor selectivity. The molecular basis of ligand selectivity of hallucinogenic and nonhallucinogenic compounds of varying structural classes for the human 5-hydroxytryptamine (5-HT)2A and 5-HT2C receptors was investigated with the use of reciprocal site-directed mutagenesis. Because these two closely related receptor subtypes differ in the amino acid present at position 5.46 (residues 242 and 222 in the sequences, respectively), the effects of corresponding substitutions in the 5-HT2A[S5.46(242)-->A] and 5-HT2C[A5.46(222)-->S] receptors were studied in tandem. By studying both receptors, the direct and indirect effects of mutations on affinity and selectivity can be distinguished. The ergolines studied, mesulergine (selective for the 5-HT2C receptor) and d-lysergic acid diethylamide (selective for the 5-HT2A receptor), reversed their relative affinity with mutations in each receptor, supporting a direct role of this locus in the selectivity of these ligands. However, interchange mutations in either receptor led to decreased or unchanged affinity for (+/-)-1-)(2,5-dimethoxy-4-iodophenyl)-2-aminopropane and ketanserin, which have higher affinity for the 5-HT2A receptor, consistent with little contribution of this locus to the selectivity of these ligands. The indoleamines studied were affected differently by mutations in each receptor, suggesting that they bind differently to the two receptor subtypes. Mutation of this locus in the 5-HT2A receptor decreased the affinity of all indoleamines, whereas the interchange mutation of the 5-HT2C receptor did not affect indoleamine affinity. These results are consistent with a direct interaction between this side chain and indoleamines for the 5-HT2A receptor but not for the 5-HT2C receptor. Furthermore, this analysis shows that the higher affinity of 5-HT and tryptamine for the 5-HT2C receptor than for the 5-HT2A receptors is not due to the difference at this locus. The hallucinogens studied [d-lysergic acid diethylamide, psilocin, bufotenin, and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane] fell into different classes in this analysis. For the classes of ligand studied, the side-chain difference at this position directly determines relative ligand selectivity only for ergolines and may contribute to the specific effects of hallucinogens in this class.

Like other amine neurotransmitters that activate G-protein-coupled receptors, 5-hydroxytryptamine (5-HT) binds to the 5-HT2A receptor through the interaction of its cationic primary amino group with the conserved Asp3.32(155) in transmembrane helix 3. Computational experiments with a 5-HT2A receptor model suggest that the same functional group of 5-hydroxytryptamine also forms a hydrogen bond with the side chain of Ser3.36(159), which is adjacent in space to Asp3.32(155). However, other 5-HT2A receptor ligands like lysergic acid diethylamide (LSD), in which the amine nitrogen is embedded in a heterocycle, or N,N-dimethyl 5-HT, in which the side chain is a tertiary amine, are found in the computational simulations to interact with the aspartate but not with the serine, due mainly to steric hindrance. The predicted difference in the interaction of various ligands in the same receptor binding pocket was tested with site-directed mutagenesis of Ser3.36(159) --> Ala and Ser3.36(159) --> Cys. The alanine substitution led to an 18-fold reduction in 5-HT affinity and the cysteine substitution to an intermediate 5-fold decrease. LSD affinity, in contrast, was unaffected by either mutation. N,N-Dimethyl 5-HT affinity was unaffected by the cysteine mutation and had a comparatively small 3-fold decrease in affinity for the alanine mutant. These findings identify a mode of ligand-receptor complexation that involves two receptor side chains interacting with the same functional group of specific serotonergic ligands. This interaction serves to orient the ligands in the binding pocket and may influence the degree of receptor activation.

The decapeptide gonadotropin-releasing hormone controls reproductive function via interaction with a heptahelical G protein-coupled receptor. Because of molecular model of the receptor predicts that Lys121 in the third transmembrane helix contributes to the binding pocket, the function of this side chain was studied by site-directed mutagenesis. Substitution of Arg at this position preserved high affinity agonist binding, whereas Gln at this position reduced binding below the limits of detection. Leu and Asp at this locus abolished both binding and detectable signal transduction. The EC50 of concentration-response curves for coupling to phosphatidyl inositol hydrolysis obtained with the Gln121 receptor was more than 3 orders of magnitude higher than that obtained for the wild-type receptor. In order to determine whether the increased EC50 obtained with this mutant reflects an altered receptor affinity, the effect of decreases in wild-type receptor density on concentration-response curves was determined by irreversible antagonism. Progressively decreasing the concentration of the wild-type receptor increased the EC50 values obtained to a maximal level of 2.4 +/- 0.2 nM. Comparison of this value with the EC50 of 282 +/- 52 nM observed with the Gln121 receptor mutant indicates that the agonist affinity for this mutant is reduced more than 100-fold. In contrast, antagonist had comparable high affinities for the wild-type, Arg121, and Gln121 mutants. The results indicate that a charge-strengthened hydrogen bond donor is required at this locus for high affinity agonist binding but not for high affinity antagonist binding.

A conserved helix 2 Asp is required for the proper function of many G-protein-coupled receptors. To reveal the structural basis for the role of this residue, the additive effects of mutations at this locus and at a conserved helix 7 locus were investigated in the 5-HT2A receptor. All mutant receptors studied retained high affinity agonist and antagonist binding. Whereas an Asp-->Asn mutation in helix 2 eliminated coupling, interchanging the residues at the two positions by a second mutation of Asn-->Asp in helix 7 restored receptor function. These data suggest that these residues are adjacent in space and interact. The loss of function observed with Ala at either position is consistent with each side chain forming hydrogen bonds. Molecular dynamics simulations were performed on three-dimensional computational models of agonist-receptor complexes of both the wild-type receptor and the Asp-->Asn mutant receptor. Consonant with the lack of coupling observed for the mutant construct, introducing the mutation into the computational model produced a conformational change in a direction opposite to that seen from computational simulations of activation of the wild-type receptor model. These results implicate both loci in a common hydrogen-bonding network underlying receptor activation by agonist.

The association of familial hypogonadism with progressive cerebellar ataxia is only rarely encountered, and the exact link between the symptoms remains unknown. We report here two sisters presenting with Holmes type cerebellar ataxia, hypogonadotropic hypogonadism and retinochoroidal degeneration recently diagnosed as Boucher-Neuhäuser syndrome. There was consanguinity between the parents of the affected individuals and the condition seemed to be inherited as an autosomal recessive defect. On endocrinological examinations, in both cases, the responses of LH and FSH to LH-RH (100 micrograms) were impaired even after repetitive stimulation with LH-RH (400 micrograms, 7 days), suggesting that the hypogonadism was due to a primary pituitary disturbance. Impaired GH responses to GRF (100 micrograms) and insulin-induced hypoglycemia (0.1 U/kg) were also noted. The two sisters shared an almost identical clinical and endocrinological picture. Their karyotypes were 46, XX. They had been treated for primary and secondary amenorrhea at the age of 20 years and neurological problems had started at the age of 30 years. This unique family displays clinical evidence of a possible common mechanism responsible for a progressive hypothalamo-pituitary and cerebellar impairment of late onset.

Prolonged exposure of the GnRH receptor to high concentrations of GnRH leads to receptor down-regulation. The role of altered receptor biosynthesis in this agonist-induced receptor down-regulation was investigated in the mouse gonadotrope cell line, alpha T3-1 cells. After exposure to 1 microM GnRH for 24 h, the number of GnRH receptor-binding sites in alpha T3-1 cells decreased to 25 +/- 6% of the control levels. No corresponding changes were observed in GnRH receptor messenger RNA (mRNA) using either quantitative ribonuclease protection/solution hybridization assay or Northern blot analysis. However, when the ability of this RNA to direct the synthesis of functional GnRH receptors was examined by quantitative assessment of the voltage clamp response in Xenopus oocytes, GnRH-induced currents in oocytes injected with RNA isolated from down-regulated cells was reduced to 40 +/- 13% of the response obtained after the injection of RNA from control alpha T3-1 cells. Thus, although GnRH receptor mRNA levels were not altered, the ability of cellular RNA isolated from alpha T3-1 cells to direct the synthesis of functional GnRH receptors was regulated in concert with receptor binding. To investigate the possibility that GnRH receptor mRNA translational efficiency was reduced, the distribution of polyribosome-associated GnRH receptor mRNA was studied. Polyribosome-associated mRNA was separated by linear sucrose gradient, and GnRH receptor mRNA distribution was determined by ribonuclease protection assay. GnRH receptor mRNA distribution shifted from the largest to smaller polyribosome and monosome fractions in cells exposed to GnRH compared to controls. The weighted mean of GnRH receptor mRNA distribution among eight fractions shifted from fraction 5.924 +/- 0.06 in control polysomes to fraction 5.45 +/- 0.219 for polysomes from down-regulated cells (P < 0.05). These results demonstrate that GnRH receptor down-regulation is accompanied by decreased GnRH receptor mRNA translation in the absence of any change in GnRH receptor mRNA levels. These data suggest that decreased efficiency of GnRH receptor mRNA translation contributes to the down-regulation of this receptor in alpha T3-1 cells.

1. The cloning of the mammalian gonadotropin-releasing hormone receptor sets the stage for rapid progress in understanding the structure of the receptor, its interaction with ligand, and its mechanisms of activation. 2. The receptor is a 327 to 328-amino acid seven-transmembrane domain G protein-coupled receptor. 3. Recent site-direct mutagenesis studies have provided considerable insight into glycosylation of the receptor, the arrangement of the helices, and the ligand binding domains.

The asparagine residues of the three N-glycosylation consensus sequences in the mouse gonadotropin-releasing hormone receptor were mutated to determine which residues were glycosylated and the function of glycosylation. Photoaffinity labelled Gln4 and Gln18 receptor mutants exhibited lower apparent molecular weight on SDS polyacrylamide gel electrophoresis, while the Gln102 receptor showed wildtype mobility. This indicates that the receptor is glycosylated at Asn4 and Asn18 but not at Asn102. Binding affinities of all the mutant receptors were normal, indicating that carbohydrate moieties are not involved in ligand binding interactions. However, expression of the Gln4 and Gln18 receptors were substantially decreased, indicating a role for glycosylation in receptor expression or stability. All the glycosylation site mutants were capable of normal signal transduction, as indicated by their ability to stimulate inositol phosphate production.

The Arg residue at position 8 of mammalian GnRH is necessary for high affinity binding to mammalian GnRH receptors. This requirement has been postulated to derive from an electrostatic interaction of Arg8 with a negatively charged receptor residue. In order to identify such a residue, 8 conserved acidic residues of the mouse GnRH receptor were mutated to isosteric Asn or Gln. Mutant receptors were tested for decreased preference for Arg8-containing ligands by ligand binding and inositol phosphate production. One of the mutants, in which the Glu301 residue was mutated to Gln, exhibited a 56-fold decrease in apparent affinity for mammalian GnRH. The mutant receptor also exhibited decreased affinity for [Lys8]GnRH, but its affinity for [Gln8]GnRH was unchanged compared with the wild type receptor. The apparent affinity of the mutant receptor for the acidic analogue, [Glu8]GnRH, was increased more than 10-fold. The mutant receptor did not, therefore, distinguish mammalian GnRH from analogues with amino acid substitutions at position 8 as effectively as the wild type receptor. This loss of discrimination was specific for the residue at position 8, because the mutant receptor did distinguish mammalian GnRH from analogues with favorable substitutions at positions 5, 6, and 7. These findings show that Glu301 of the GnRH receptor plays a role in receptor recognition of Arg8 in the ligand and are consistent with an electrostatic interaction between these 2 residues.

The mouse gonadotropin-releasing hormone receptor (GnRHR) is unique among G-protein-coupled receptors in its lack of a putative intracellular carboxy-terminal domain. A gonadotrope cell line cDNA library was screened in a search for alternative forms of the receptor transcript and 42 clones were obtained, representing a number of variant cDNAs. To determine the origin of these transcripts, the structure of the mouse gene was mapped from 11 distinct genomic clones. The gene contains three exons, spanning more than 22 kb. Exons 1, 2, and 3 encode, respectively, nucleotides +1 to +522, +523 to +739, and +740 to +981 of the open reading frame of the cDNA for the functional mouse GnRHR. Southern blot analysis with genomic DNA is consistent with the presence of a single gene. By comparison with the genomic sequence, the origins of the variant cDNAs isolated can be clarified. All the cDNAs contain the first exon and the majority (71%) encode the functional 327-amino-acid receptor previously reported. One group of clones (14%), which contains exons 1 and 2, continues 700 bp past the exon 2 splice donor of the wild-type receptor. These clones terminate after a polyadenylation signal and have an open reading frame encoding a protein of only 261 amino acids. In a different group of transcripts (5%), exon 2 is absent, resulting in a shift in the reading frame and encoding a protein of 177 amino acids. These data support alternative processing of the mouse GnRHR gene.

GnRH regulates the synthesis and secretion of pituitary gonadotropins. The number of receptors for GnRH (GnRH-rec) can vary from 500 to 15,000-20,000/gonadotrope in ovine pituitary cultures after treatment with physiologically relevant combinations of gonadal hormones. This large range suggests that regulation of GnRH-rec expression may be an important control point in GnRH action at the pituitary level. Reported here are the changes in GnRH-rec mRNA associated with pituitary treatments (48 h) of 17 beta-estradiol (E), progesterone (P), and an enriched preparation of porcine follicular inhibin (IN). Northern blot analysis was used to detect 3 species of GnRH-rec mRNA in primary ovine pituitary culture [5.5 kilobases (kb; 32%), 3.6 kb (51%), and 1.4 kb (17%)]; all were changed in parallel by E, P, and IN. GnRH-rec mRNAs were increased 190% over control levels after treatment with either E or IN, and 400% with E and IN combined; when E and IN were added along with P, the increase was only 50% (P caused an 87% inhibition of E plus IN induction). The addition of P in the absence of any other treatment reduced levels of GnRH-rec mRNA by 50%. Studies were also conducted with GnRH agonists (GnRH-A) due to their widespread clinical use for down-regulating reproductive function in men and women. The addition of GnRH-A to cultures was as effective as P in blocking E plus IN induction of GnRH-rec mRNA. In vivo studies in wethers showed that 7 days of chronic treatment with GnRH-A decreased all sizes of ovine GnRH-rec mRNA by 84-89%. These data indicate that E, P, and IN change GnRH-rec levels at least in part by changing levels of GnRH-rec mRNAs. They also show that GnRH-A can almost entirely block E plus IN induction of GnRH-rec mRNA in vitro and decrease levels of GnRH-rec mRNA in vivo in wethers.

Activation of the pituitary gonadotropin-releasing hormone receptor, a member of the seven-transmembrane G protein-coupled receptor (GPCR) family, triggers a cascade of events leading to gonadotropin release and stimulation of the reproductive system. An unusual feature of this receptor, observed in mice, rats, and humans, is the presence of Asn87 in the second putative transmembrane helix at the location of a highly conserved aspartate in the GPCR family and of Asp318 in the putative seventh transmembrane helix where nearly all other GPCRs have asparagine. The possibility that these residues interact was suggested by this reciprocal pattern and by a three-dimensional model of the gonadotropin-releasing hormone receptor and was investigated by site-directed mutagenesis. Replacing Asn87 in the second transmembrane domain by aspartate eliminated detectable ligand binding. A second mutation, generating the double-mutant receptor Asp87Asn318, recreated the arrangement found in other GPCRs and re-established high affinity agonist and antagonist binding. The restoration of binding by a reciprocal mutation indicates that these two specific residues in helices 2 and 7 are adjacent in space and provides an empirical basis to refine the model of the transmembrane helix bundle of the receptor.

In primary cultures of rat pituitary, GnRH receptor has been reported to up-regulate when exposed to GnRH. The molecular basis of this regulation could, in theory, involve modulation of gene transcription, RNA processing or stability, translation, and/or receptor degradation. The role of altered biosynthesis was investigated in the mouse gonadotrope cell line, alpha T3-1 cells, by studying the homologous up-regulation of GnRH receptor binding, mRNA levels, and mRNA activity. After GnRH exposure, ligand binding was performed on alpha T3-1 cell membranes. To correlate changes in receptor number and measurements of biosynthetic activity, GnRH receptor mRNA levels were quantitated by solution hybridization/RNase protection assay and Northern blot analysis, and the capacity of RNA from treated cells to direct the synthesis of new receptors (mRNA activity) was evaluated using a Xenopus oocyte-based bioassay. GnRH agonist radioligand binding assay results showed that exposure of alpha T3-1 cells to 10(-10) or 10(-8) M GnRH for 20 min induced an approximately 50% increase in the number of GnRH receptors, similar to previously reported results in rat pituitary primary culture. Despite the increase in receptor number, however, cytosolic and total GnRH receptor mRNA levels assayed by solution hybridization/RNase protection assay with GnRH receptor cRNA probes and by Northern blot analysis were not altered. In contrast to the unchanging mRNA levels, the measurements of mRNA activity paralleled the changes observed at the binding level. alpha T3-1 RNA from treated and control cells were injected into Xenopus oocytes, and the GnRH-induced Cl- current was quantified 48 h later by voltage clamp recording of the response to GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)

The cloned sheep gonadotropin-releasing hormone (GnRH) receptor was analysed for sequence homology/differences among mammalian receptors and its pharmacology characterized in COS-1 cells. Transmembrane domains TM2, TM3, TM5, TM6 and TM7, and extracellular loop 1 are most highly conserved (> 90%) in this G-protein coupled receptor. The Kd of the sheep receptor in binding assays (4.9 nM) was similar to the human and rat receptors, but lower than the mouse receptor. The rank order of potency of a series of GnRH analogues for binding and inositol phosphate stimulation in transfected COS-1 cells was identical to that of the receptor characterized in sheep pituitary gonadotropes. Northern blot analysis identified four transcripts sized 5.4 kb, 3.6 kb, 2.3 kb and 1.3 kb in sheep pituitaries which were upregulated by castration.

The influence of acute and chronic cocaine treatment on the expression of dopamine D2 receptor (D2R) mRNA was examined in the rat basal ganglia by in situ hybridization histochemistry. No significant alterations in D2R mRNA levels were observed in the striatum or the nucleus accumbens following acute or chronic cocaine. In the olfactory tubercle, acute cocaine (20 and 40 mg/kg, i.p.) was found to decrease D2R mRNA expression. Chronic cocaine (20 mg/kg, i.p. x 15 days), however, produced an increase in D2R mRNA levels in this region which was detected 24 h, but not 7 days after withdrawal. Acute and chronic cocaine have different effects on the regulation of D2R gene expression in the olfactory tubercle.

The primate striatum has a compartmental organization reflected both in the topography of its afferent projections and in the segregation of its morphologically similar but neurochemically distinct efferent neurons. Discretely projecting mesostriatal neurons release dopamine (DA) which modulates the responses of striatal neurons to other afferent inputs. Multiple DA receptor (DAR) subtypes have been cloned and characterized and mapping their cellular expression is crucial for understanding the influence of DA on striatal function. We report the distribution of mRNAs for D1, D2 and D5 DAR subtypes (D2R, D2R and D5R) in the striatum of cynomolgus monkeys (Macaca fascicularis) studied by in situ hybridization histochemistry (ISH) using monkey-specific cRNA probes. Adjacent sections were stained for calbindin immunoreactivity to distinguish striosomal and matrix compartments for comparison with the patterns obtained with ISH. In the caudate nucleus, D1R mRNA was concentrated in calbindin-poor striosomes where dense grain clusters were seen overlying the majority of medium-sized neurons (diameter approximately 15 microns). D1R mRNA localization was relatively homogeneous in the putamen. By contrast, the distributions of D2R and D5R mRNAs showed no clear preference for the striosomal or matrix compartments of either caudate nucleus or putamen. In the ventral striatum (nucleus accumbens, olfactory tubercle and ventral portions of caudate nucleus and putamen), expression of D1R and D2R mRNA was sparse relative to dorsal striatum, while D5R mRNA expression was roughly equal in ventral and dorsal striatum. Circumscribed zones of hybridization associated with islands of tightly packed small cells occurred with all three DAR mRNA subtypes in the ventral striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

A cDNA encoding the human GnRH receptor (GnRHR) has been cloned and functionally expressed in both Xenopus oocytes and COS-1 cells. The 2160 bp cDNA encodes a 328 amino acid protein with a predicted amino acid sequence that is 90% identical to that of the mouse GnRHR (Tsutsumi et al. (1992) Mol. Endocrinol. 6, 1163-1169). Injection of synthetic RNA transcript into oocytes led to the development of a depolarizing response to agonists when assayed by voltage-clamp electrophysiology. Consistent with the expression of a mammalian GnRHR, the response was blocked by GnRH antagonists. Following expression of the human GnRHR in COS-1 cells, agonists and an antagonist displaced [125I]GnRH agonist from membrane isolates with nanomolar range dissociation constants similar to those described for displacement from human pituitary membranes. Transfected COS-1 cells manifested a GnRH-stimulated increase in phosphoinositol turnover, with an EC50 of approximately 3 nM, which was inhibited by GnRH antagonists. Northern blot analysis revealed a single band of approximately 4.7 kb expressed in human pituitary which was not detected in testis. The predicted structure of the human GnRHR is similar to that previously reported for the mouse receptor. Although the mammalian GnRHR is a seven transmembrane domain receptor, it differs from other G-protein coupled receptors in several respects, most notably the lack of a cytoplasmic C-terminal domain. The present study demonstrates that the cDNA isolated encodes the human GnRHR and suggests that several unique features conserved among mammalian GnRHRs may be essential for receptor function and/or regulatory control.

Dopamine plays a critical role in motor and cognitive function through actions mediated by specific receptors, multiple subtypes of which have recently been identified. The distribution of mRNAs encoding D1, D2 and D5 receptors in the motor cortex of humans and in the motor cortex and striatum of macaque monkeys was examined using in situ hybridization. In motor cortices from both primate species, hybridization to each receptor probe resulted in numerous labeled cells throughout layers II-VI. In contrast to neocortex, in monkey striatum only the D1 and D2 receptor probes showed significant hybridization. Thus, not only does primate neocortex possess a broader representation of the dopamine receptor subtype mRNAs examined in comparison with striatum, but the unexpected presence and widespread distribution of D2 and D5 receptor mRNAs in cortex suggests that, along with D1 receptors, D2 and D5 receptors play a crucial role in the dopaminergic modulation of cognition and motor behavior, and in dopamine dysfunction associated with neuropsychiatric disorders.

GnRH plays a pivotal role in the reproductive system, and GnRH analogs have wide therapeutic applications ranging from the treatment of prostatic cancer to infertility. Determination of the predicted structure of the GnRH receptor (GnRHR) would illuminate the mechanisms of receptor activation and regulation and allow directed design of improved GnRH analogs. We report the cloning of a cDNA representing the mouse GnRHR and confirm its identity using Xenopus oocyte expression. Injection of sense RNA transcript leads to the expression of a functional, high affinity GnRHR. Expression of the GnRHR using gonadotrope cell line RNA, however, is blocked by an antisense oligonucleotide. In situ hybridization in the rat anterior pituitary reveals a characteristic GnRHR distribution. The nucleotide sequence encodes a 327-amino acid protein which has the seven putative transmembrane domains characteristic of G protein-coupled receptors, but which lacks a typical intracellular C-terminus. The unusual structure and novel potential regulatory domain of the GnRHR may explain unique aspects of its signal transduction and regulation.

The multitude of G-protein coupled receptor (GPR) superfamily cDNAs recently isolated has exceeded the number of receptor subtypes anticipated by pharmacological studies. Analysis of the sequence similarities and unique features of the members of this family is valuable for designing strategies to isolate related cDNAs, for developing hypotheses concerning substrate-ligand and receptor-effector interactions, and for understanding the evolution of these genes. We have compiled and aligned the 74 unique amino acid sequences published to date and review the present understanding of the structural motifs contributing to ligand binding and G-protein coupling.

A cDNA for the rat dopamine D3 receptor containing a 113 bp deletion has been isolated. The segment deleted, encompassing the first extracellular loop and third transmembrane domain, alters the reading frame, introducing 19 amino acids not found in the full length receptor followed by a premature stop codon. This novel mRNA encodes a 109 amino acid protein containing two putative transmembrane domains. A similar variant cDNA for the human D3 receptor has also been identified.

Hypophyseal portal dopamine is a major negative regulator of pituitary prolactin (PRL) production. Dopamine has been reported to repress PRL gene transcription in pituitary cells. To facilitate further study of the effect of dopamine on PRL gene activation, we introduced PRL promoter and D2 receptor (D2R) constructs into GH3 cells. Since two D2R isoforms (termed D2S and D2L) have been cloned previously, we first determined which isoform(s) is present in the lactotroph by measuring the level of each mRNA species in rat prolactinoma. mRNA for each D2R isoform was found to be present, with the D2L mRNA in great (c. 6-fold) excess. Because the lactotroph contains both isoforms, the effect of each on the PRL promoter was investigated. The cDNA for each receptor isoform was synthesized by polymerase chain reaction, and cloned into an RSV-based expression vector. GH3 cells were then transiently co-transfected with either of the resulting RSV-D2R constructs plus a PRL-chloramphenicol acetyltransferase (CAT) construct containing the first 1957 base-pairs of PRL gene 5'-flanking DNA. The cells were then incubated 48 h plus or minus the dopamine agonist ergocryptine (ECR). In the presence of either RSV-D2R isoform, ECR yielded a 4-5-fold decrease in CAT activity, an effect not seen in the absence of the RSV-D2R. The promoter specificity of this effect was demonstrated by the inability of ECR to regulate expression of a control RSV-CAT construct. The PRL promoter repression mediated by each receptor isoform had appropriate pharmacology: the specific D2R agonist, quinpirole, yielded results similar to ECR, and the ECR repression was reversed by the dopamine antagonist spiperone.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated the distribution of the two dopamine D2 receptor mRNA splice variants in the rat using a sensitive and quantitative solution hybridization/nuclease protection assay. In all brain and endocrine regions studied, both splice variants were detected and the mRNA of the longer form (D2L) was more abundant than that of the shorter form (D2S). The lowest percentages of D2S were found in the pituitary and adrenal glands.

Previous studies demonstrate that gonadotroph responsiveness to GnRH, GnRH binding, and the apparent number of GnRH receptors are all increased by 17 beta-estradiol (E) or inhibin (IN) in ovine pituitary cultures. Progesterone (P) attenuates these effects. To explore differences between the effects of IN and E on GnRH binding, a detailed time-course study was performed. The results indicate that after 48 h, IN had a greater effect on binding of a GnRH agonist (5-fold increase) than E (3-fold increase), but was slower to act initially. A combined treatment of IN and E gave a partially additive effect at 48 h (6.5-fold increase). The mechanism of receptor regulation in this system is not known, but could involve synthesis, recycling, or modification of GnRH receptors. To investigate the contribution of altered receptor biosynthesis to the regulation of receptor levels, a functional Xenopus oocyte-based assay for GnRH receptor mRNA activity was employed. After 48 h of treatment, IN or E each led to a 7- to 8-fold increase in GnRH receptor mRNA activity. Treatment with both hormones led to a 19-fold increase. The increase in mRNA activity induced by either hormone was greatly attenuated by P. Modulation of GnRH receptor mRNA levels suggests that IN, E, and P regulate responsiveness to GnRH in the ovine pituitary at least in part by altering de novo synthesis of GnRH receptors. The differing time courses of action, as assayed by GnRH binding, and the additivity of effects at the mRNA level suggest that IN and E alter mRNA levels via different mechanisms.

We investigated the effect of intracellular loading of the non-hydrolyzable guanosine 5'-diphosphate analogue GDP beta S on calcium mobilization by IP3 and on calcium influx in Xenopus oocytes. Assayed by two electrode voltage-clamp recording, GDP beta S-loaded oocytes demonstrated a marked augmentation of the fast component of the response to IP3 injection, an attenuation of the slow component and an increase in membrane calcium permeability. These effects on calcium mobilization suggest that GDP beta S may facilitate calcium translocation both across the plasma membrane and between different intracellular calcium pools.

The rodent GnRH receptor was characterized in Xenopus oocytes injected with RNA isolated from rat pituitary and from a gonadotrope cell line, alpha T3, derived from a transgenic mouse. Three to 4 days after 150-200 ng RNA injection, 93% of the oocytes, which were recorded by voltage clamp, responded to 10(-7) M GnRH. The mean inward currents obtained after RNA injection were 620 +/- 88 nA (n = 22) with pituitary RNA and 1415 +/- 598 (n = 4) with alpha T3 RNA. The threshold GnRH concentration able to evoke the dose dependent current after pituitary RNA injection was 3 x 10(-9) M GnRH. The GnRH receptor response of the oocyte was antagonized by [D-Phe2,6,Pro3] GnRH and [N-Ac-D-Na](2)1, D-alpha D-Me, pCl-Phe2, D-Arg6, D-Ala10-NH2]GnRH and could be elicited by D-Ser(But)6,Pro9-N-ethylamide GnRH (buserelin). The reversal potential of the GnRH generated current as determined by voltage-ramp was -22.5 +/- 1.0 mV (n = 7) and -25.6 +/- 3.3 mV (n = 3) in pituitary and cell line RNA-injected oocytes respectively, consistent with the chloride reversal potential. The GnRH receptor response was virtually eliminated by intracellular EGTA injection but was unaffected by ligand application in calcium-free perfusate. The GnRH-evoked response is mimicked by intracellular injection of inositol 1,4,5-trisphosphate. To determine the size of the GnRH receptor mRNA, alpha T3 RNA was size fractionated through a sucrose gradient. The maximal GnRH response was induced by a fraction larger than the 28S ribosomal peak. Thus we find that oocytes injected with RNA from an appropriate source develop an electrophysiological response to GnRH which is dependent on intracellular calcium mobilization, is independent of extracellular calcium, and may be mediated by inositol 1,4,5-trisphosphate.

Heterotrimeric guanine nucleotide-binding proteins (G-proteins) can be categorized into molecularly divergent groups by their differential sensitivity to pertussis toxin. Receptors specifically use either pertussis toxin-sensitive or-insensitive G-proteins to couple to specific effectors. Receptor stimulation of phospholipase C, however, is pertussis toxin sensitive in some systems and pertussis toxin insensitive in others. We studied the coupling of receptors to phospholipase C by expressing receptors from both systems into a single cell, the Xenopus oocyte. [Arg8]Vassopressin (AVP) receptors from liver and cholecystokinin-8(sulfated) (CCK) receptors from brain were expressed in oocytes by intracellular injection of RNA. Both receptors stimulated a Ca2+-dependent Cl- current which can also be evoked by intracellular injection of inositol 1,4,5-tris-phosphate. Hence, receptor stimulation of phospholipase C was measured as the evoked Ca2+-dependent Cl- current. The liver AVP receptor, which is known to stimulate phospholipase C in a pertussis toxin-insensitive manner (Lynch, C. J., Prpic, V., Blackmore, P. F., and Exton, J. H. (1986) Mol. Pharmacol. 29, 196-203), was found to stimulate phospholipase C through a pertussis toxin-sensitive pathway in the Xenopus oocyte. The CCK receptor from brain stimulated phospholipase C through a pertussis toxin-insensitive pathway. Both AVP and CCK stimulation of phospholipase C were attenuated by the intracellular injection of excess G-protein beta gamma subunits. Neither pertussis toxin treatment nor intracellular injection of beta gamma subunits affected any steps subsequent to inositol 1,4,5-tris-phosphate production. From these data we conclude that both the pertussis toxin-sensitive and -insensitive pathways for receptor coupling to phospholipase C are transduced by heterotrimeric G-proteins. We also find that there is a lack of coupling fidelity of receptors to G-proteins in stimulation of phospholipase C which can be influenced by the membrane environment.

Hypothalamic dopamine (DA), acting at DA D2-receptors (D2-R) on pituitary target cells, mediates peptide release and biosynthesis of rat pituitary anterior lobe (AL) prolactin, and neurointermediate lobe (NIL) pro-opiomelanocortin (POMC). We were interested in determining if dopamine agonists and antagonists were capable of modifying D2-R gene expression in these pituitary cells. Utilizing the recently published sequence of the rat D2-R, we isolated a rat D2-R cDNA clone by polymerase chain reaction, and have synthesized RNA probes to quantitate levels of D2-R mRNA by solution hybridization/nuclease protection assay. We report here that 5-day administration of the DA antagonist haloperidol led to significant increases in both D2-R mRNA and POMC mRNA in the NIL; the DA agonist bromocriptine caused a significant decrease in NIL POMC mRNA with no parallel change in D2-R mRNA. In contrast, no significant changes in D2-R mRNA in AL were observed following treatment with either the DA agonist or antagonist. These data provide evidence for tissue-specific regulation of D2-R mRNA in response to dopaminergic manipulation.

1. Membrane currents were studied in voltage-clamped Xenopus laevis oocytes which had been injected with total rat brain RNA. 2. When the membrane potential was stepped from -100 to +10 mV, two components of outward current were observed which were named Tout1 and Tout2. 3. Both Tout1 and Tout2 were eliminated in chloride-free or calcium-free media and blocked by 9-anthroic acid, indicating that they represented calcium-dependent chloride currents. 4. Both currents were dependent on extracellular calcium (1.8-10 mM), with Tout1 showing a greater sensitivity to changes in calcium concentration. 5. Tout2 but not Tout1 was blocked by intracellular injection of 300-600 pmol, BaCl2 (final concentration in the oocyte: 0.3-0.6 mM). Injection of KCl had no effect on either Tout1 or Tout2. 6. Tout2 but not Tout1 was enhanced by low concentrations of serotonin (0.5-2 nM). This effect was blocked by 0.1 microM-mianserin. Higher concentrations (above 10 nM) of serotonin decreased the amplitude of Tout2. The effect of serotonin was blocked by the protein kinase inhibitor, H-7 (25 microM). 7. Tout2 but not Tout1 was enhanced by 10 nM-phorbol myristate acetate. Higher concentrations of the phorbol ester decreased the amplitude of Tout2. 8. It is concluded that in oocytes injected with RNA there is an induction of a novel component of the calcium-induced chloride current (Tout2). This current reflects a second process of chloride channel opening which can be enhanced by serotonin via activation of protein kinase C.