Molecular pharmacology of NMDA receptors: modulatory role of NR2 subunits.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
P B Molinoff
Publications and source records attributed to P B Molinoff.
International Union of Pharmacology nomenclature of adrenoceptors.
Explore the source record for details and available documents.
Adrenergic Agonists↗
Mechanisms of up-regulation of D2L dopamine receptors by agonists and antagonists in transfected HEK-293 cells.
Mechanisms underlying agonist- and antagonist-induced up-regulation in HEK-293 cells transfected to express D2 dopamine receptors were investigated. We have reported previously that exposure of cells to agonists and antagonists led to an increase in the density of receptors. The time course of up-regulation on exposure to (-)-sulpiride, a D2 dopamine-selective antagonist, was measured. A lag in the effect of antagonists not seen with up-regulation induced by exposure to agonists was observed. Effects of N-propylnorapomorphine were synergistic with those of cyclic AMP analogs, however, synergistic effects between (-)-sulpiride and cyclic AMP analogs were not observed. These findings suggest that separate mechanisms may be involved in agonist- and antagonist-induced up-regulation. Changes in mRNA levels did not appear to account for the increase in receptors after agonist or antagonist treatment. Results of experiments with cycloheximide, a protein-synthesis inhibitor, suggest that increased protein synthesis, and not decreased protein degradation, is responsible for up-regulation by both NPA and (-)-sulpiride. Studies monitoring receptor recovery after treatment with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, an irreversible alkylating agent, suggest that rates of receptor incorporation into membranes are increased after treatment with either an agonist or an antagonist.
Apomorphine↗
Characterization of [125I](R)-trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)amino] tetralin binding to dopamine D3 receptors in rat olfactory tubercle.
[125I](R,S)-trans-7-Hydroxy-2-[N-propyl-N-(3'-iodo-2'-propenyl)- amino]tetralin ([125](R,S)-trans-7-OH-PIPAT) has been shown to bind with high affinity to dopamine D3 receptors expressed in Spodoptera frugiperda cells. No specific binding was seen in Spodoptera frugiperda cells expressing a high density of D2 receptors. It was therefore, suggested that [125I] (R,S)-trans-7-OH-PIPAT selectively labels D3 receptors. In the present study, saturation binding of [125I](R)-trans-7-OH-PIPAT to membranes from rat olfactory tubercle resulted in markedly curvilinear Scatchard plots, suggesting that the radioligand was binding to more than one receptor class or affinity state. [125I] (R)-trans-7-OH-PIPAT bound with high affinity to membranes from human embryonic kidney-293 cells expressing transfected D2 or D3 receptors and to membranes from Chinese hamster ovary cells expressing serotonin1A receptors. Binding of [125I](R)-trans-7-OH-PIPAT to serotonin1A and D2 receptors was decreased or eliminated in the presence of NaCl and/or guanylyl-imidodiphosphate [Gpp(NH)p]. In the presence of Gpp(NH)p and NaCl, a linear Scatchard plot with a Kd value of 0.4 nM and a density of 100 fmol/mg of protein was obtained in membranes from rat olfactory tubercle. Agonists and antagonists inhibited binding of [125I](R)-trans-7-OH-PIPAT with a rank order of potency consistent with an interaction at D3 receptors. These results suggest that, in the presence of Gpp(NH)p and NaCl, [125I](R)-trans-7-OH-PIPAT specifically labels D3 receptors.
Animals↗
Characterization of a 5-hydroxytryptamine receptor in mouse neuroblastoma N18TG2 cells.
Cyclic AMP formation was found to increase in mouse neuroblastoma N18TG2 cells exposed to 5-hydroxytryptamine (5-HT). This response was concentration-dependent with an EC50 value of 0.22 microM. Tryptamine and other tryptamine-related compounds were also agonists in this assay with a rank order of potency of 5-methoxytryptamine > 5-HT > tryptamine > 2-methyl-5-HT > 5-carboxamidotryptamine >> alpha-methyl-5-HT. (D)-Lysergic acid diethylamide and 2-bromo-lysergic acid diethylamide were partial agonists in this system with maximal responses of 44 and 34%, respectively, compared to 5-HT. 5-HT-stimulated cyclic AMP formation was inhibited by clozapine, mianserin and methiothepin with pA2 values of 6.6, 6.5 and 6.4, respectively. Radioligand binding studies using [125I]iodolysergic acid diethylamide revealed a binding site present at a density of 10.4 fmol/mg of protein, with an affinity for the ligand of 1.18 nM. In competition studies this binding site displayed a pharmacology similar to that defined in studies of cyclic AMP formation. The pharmacological profile of this receptor, characterized by both radioligand binding and functional coupling to adenylyl cyclase, does not correspond to that of any of the currently classified subtypes of 5-HT receptor, but is similar to the 5-HT receptor cloned recently from rat striatum and referred to as the 5-HT6 receptor. The N18TG2 cell line represents a useful model system in which this novel 5-HT receptor may be characterized fully.
Adenylyl Cyclases↗
Sensitivity of the N-methyl-D-aspartate receptor to polyamines is controlled by NR2 subunits.
The endogenous polyamine spermine has multiple effects on the N-methyl-D-aspartate (NMDA) receptor. These include an increase in the magnitude of NMDA-induced whole-cell currents that is seen in the presence of saturating concentrations of glycine ("glycine-independent" stimulation), an increase in the affinity of the receptor for glycine ("glycine-dependent" stimulation), and voltage-dependent inhibition. Although many of the properties of native NMDA receptors are seen with homomeric NR1 receptors expressed in Xenopus oocytes, we have found that the effects of spermine are differentially regulated by NR2 subunits in heteromeric NR1/NR2 receptors. Glycine-independent stimulation by spermine occurred at homomeric NR1A receptors, which lack the amino-terminal insert in NR1, and at heteromeric NR1A/NR2B receptors but not at heteromeric NR1A/NR2A or NR1A/NR2C receptors. Glycine-independent stimulation was not seen at homomeric NR1B receptors, which include the amino-terminal insert in NR1, or at heteromeric receptors containing NR1B. Thus, glycine-independent stimulation by polyamines requires the presence of an NR1 variant, such as NR1A, that lacks the amino-terminal insert, but the manifestation of the stimulatory effect is controlled by the type of NR2 subunit present in a heteromeric complex. Glycine-dependent stimulation was seen at NR1A/NR2A and NR1A/NR2B receptors and may therefore involve a second polyamine binding site distinct from that which produces glycine-independent stimulation. The voltage-dependent inhibitory effect of spermine, which is more pronounced at hyperpolarized membrane potentials, occurred with similar magnitudes at NR1A/NR2A and NR1A/NR2B receptors but was absent at NR1A/NR2C receptors. Thus, NR2 subunits control both the stimulatory and inhibitory effects of spermine at NMDA receptors. Stimulation but not inhibition by spermine was seen at NR1A/NR2B receptors in the presence of extracellular Mg2+. Stimulation, seen in the presence of physiological concentrations of Ca2+ and Mg2+, may be the predominant effect of polyamines at NMDA receptors in the intact nervous system.
Animals↗
Expression of mRNAs encoding subunits of the N-methyl-D-aspartate receptor in cultured cortical neurons.
The expression of mRNAs encoding subunits of the N-methyl-D-aspartate (NMDA) receptor was examined in cortical neurons maintained in primary culture. Cultures were prepared from embryonic day 17 rat neocortex. At this developmental age, levels of NR1, NR2A, NR2B, and NR2C mRNA were low or undetectable. Expression of NR1 mRNA increased progressively between days 1 and 21 in vitro. The amount of NR2A mRNA did not change between days 1 and 7 but increased between days 7 and 21. In contrast, levels of NR2B mRNA increased between days 1 and 7, with little further change after day 7. The level of NR2B mRNA was approximately 4-fold higher than that of NR2A mRNA in 21-day cultures. Using ligand binding assays, the proportion of NMDA receptors having a low affinity for ifenprodil was also found to increase over time in culture. The increase in the expression of receptors having a low affinity for ifenprodil and the increase in NR1 and NR2A mRNAs were reduced or prevented by maintaining cells in medium with a low concentration of serum. The results are consistent with the hypothesis that inclusion of the NR2A subunit in native NMDA receptors is responsible for their low affinity for ifenprodil. Splice variants of NR1 lacking the 5' (amino-terminal) insert were found to be the predominant forms of NR1 in cultured neurons. Variants containing the 5' insert represented only a small (< or = 5%) fraction of total NR1 mRNA, and their proportion was not altered as a function of time in culture. Time-dependent changes in the properties of NMDA receptors and in the expression of subunit mRNA occurring in cultured neurons are similar to changes observed in developing rat brain. Thus, the developmental sequence of NMDA receptor expression that occurs in vivo is partially retained in neurons maintained in vitro.
Animals↗
Developmental switch in the expression of NMDA receptors occurs in vivo and in vitro.
The properties of many ligand-gated ion channels are altered during development. We have characterized a developmental switch in the sensitivity of NMDA receptors to the novel antagonist ifenprodil using ligand binding assays with rat brain membranes and voltage-clamp recording of Xenopus oocytes expressing NMDA receptors after injection of RNA from rat brain and from cloned subunits of the receptor. In neonatal rat brain, NMDA receptors have a uniformly high affinity for ifenprodil. During postnatal development, a second population of receptors having a 100-fold lower affinity for ifenprodil is expressed and represents 50% of NMDA receptors in adult rat brain. This developmental change also occurred in cortical neurons maintained in primary culture. Ifenprodil potently inhibited responses of homomeric NR1 and heteromeric NR1/NR2B receptors but not NR1/NR2A receptors expressed in oocytes, suggesting that inclusion of different NR2 subunits in native NMDA receptors can control the sensitivity to ifenprodil.
Animals↗
Development of polyclonal anti-D2 dopamine receptor antibodies to fusion proteins: inhibition of D2 receptor-G protein interaction.
Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins. The fusion proteins were gel-purified and used to immunize rabbits for the production of polyclonal anti-receptor antisera. The anti-fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid-phase radioimmunoassay. Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist [125I]NCQ 298 and digitonin-solubilized extracts of canine and rat caudate. [125I]-NCQ 298 bound reversibly and with high affinity (KD = 0.14 nM) to receptors in solubilized extracts enriched by chromatography on heparin-agarose. The anti-UBI-D2i3L and anti-UBI-D2i3s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with [125I]NCQ 298 from solubilized rat caudate. The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP-binding proteins in digitonin-solubilized rat caudate. Both anti-UBI-D2i3L and anti-UBI-Di3s antisera were able to inhibit the high-affinity binding of the agonist N-propylnorapomorphine to digitonin-solubilized rat caudate. These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein.
Amino Acid Sequence↗
Paradoxical regulation of dopamine receptors in transfected 293 cells.
Selective expression of subtypes of receptors in mammalian cell lines permits the study of the regulation of receptors in a homogeneous population of cells growing under controlled conditions. cDNAs encoding the human D2L and D2S receptors were ligated into a eukaryotic expression vector, pRc/CMV. The resulting plasmid, which contains a cytomegalovirus promoter for high expression levels, was used for stable transfection of 293 cells, a human kidney cell line. Expression of D2L and D2S receptors in 293 cells was confirmed by radioligand binding assays with [125I]NCQ 298. The pharmacological properties of the expressed receptors were comparable to those of receptors in rat striatal homogenates and in other transfected cell lines. D2L and D2S receptors were coupled to inhibition of cAMP accumulation in 293 cells. Incubation of 293-D2L cells with agonists resulted in an increase in the density of D2 receptors without a change in the affinity of the receptors for [125I]NCQ 298. This effect was time dependent, with a t1/2 of approximately 6 hr. The dose dependence of up-regulation followed the pharmacological profile expected of a D2 receptor, with an order of potency of N-propylnorapomorphine (NPA) > quinpirole > dopamine. The density of receptors was further increased by incubation of cells with agonist together with forskolin or 8-bromo-cAMP. D2S receptors responded similarly to D2L receptors to treatment with NPA and forskolin. Exposure of 293-D2L cells to the beta-adrenergic receptor agonist isoproterenol did not change the density of D2L receptors. Similarly, NPA had no effect on levels of endogenously expressed beta-adrenergic receptors in 293-D2L cells, as assayed by binding of [125I]iodocyanopindolol. Levels of beta-adrenergic receptors in transfected 293-beta 2 or 293-D2L cells did not increase after exposure to NPA but decreased after exposure to isoproterenol. Cells expressing D2L receptors were incubated with antagonists, including SCH-23390, sulpiride, haloperidol, clozapine, and epidepride, alone or in combination with NPA. Incubation of cells with SCH-23390 had no effect on the density of D2 receptors, and SCH-23390 did not block the effect of NPA. D2-selective antagonists increased the density of receptors. D2L receptor mRNA levels were unchanged during agonist treatment. This suggests a role for translational or post-translational mechanisms in the regulation of D2 receptor levels in transfected cell lines.
8-Bromo Cyclic Adenosine Monophosphate↗
Effects of agonists, partial agonists, and antagonists on the regulation of 5-hydroxytryptamine2 receptors in P11 cells.
Studies of the regulation of 5-hydroxytryptamine (5-HT2) receptors in vivo have generated anomalous and sometimes contradictory results. In particular, administration of antagonists unexpectedly results in a reduction in the density of 5-HT2 receptors. P11 cells, which express a high density of 5-HT2 receptors coupled to phosphoinositide hydrolysis, were used to investigate the regulation of receptors in vitro by agonists, partial agonists, and antagonists. (+/-)-2,5-Dimethoxy-4-iodophenylisopropylamine (DOI) and (+)-lysergic acid diethylamide (LSD) caused marked reductions in the density of 5-HT2 receptors as has been observed in vivo. Down-regulation was prevented by coincubation with ketanserin. The decrease in the density of 5-HT2 receptors after exposure to 5-HT, LSD, or DOI was time dependent and was not a consequence of residual drug in binding assays or irreversibly bound drug. The ability of 5-HT, DOI, and LSD to down-regulate 5-HT2 receptors was not proportional to the ability of these compounds to stimulate phosphoinositide hydrolysis. Ketanserin and mianserin, antagonists which cause paradoxical decreases in the density of 5-HT2 receptors in vivo, did not alter the density of 5-HT2 receptors on P11 cells, even after prolonged incubation with drug. Results of the current studies, which demonstrate agonist- but not antagonist-induced down-regulation of 5-HT2 receptors, lead to the conclusion that the ability of ketanserin and mianserin to down-regulate receptors in vivo is the result of indirect actions of these drugs and is unlikely to be a direct consequence of receptor occupancy by antagonists.
Amphetamines↗
Expression and characterization of the rat D3 dopamine receptor: pharmacologic properties and development of antibodies.
A baculovirus expression system provided an enriched source of biologically and immunologically active D3 dopamine receptors. Receptors expressed in Spodoptera frugiperda insect (Sf9) cells at a density of 5 to 15 pmol/mg of protein displayed high affinity for the antagonists, eticlopride, fluphenazine and spiroperidol, and the agonist, N-propylnorapomorphine. The binding of agonists was not sensitive to GTP. Antisera raised against synthetic peptides in the third intracellular loop of the D3 dopamine receptor immunoprecipitated binding sites for (S)-3-[125I]-iodo-2-hydroxy-5,6-dimethoxy-N-[(1-ethyl-2-pyrrolidinyl)- methyl]-benzamide from solubilized extracts of infected Sf9 cells and detergent extracts of rat caudate. These antisera specifically recognized a single band on immunoblots of Sf9 cells infected with recombinant D3 baculovirus. Both the immunoprecipitation and immunoblot reactions were blocked by preincubation of the antisera with the immunization peptide. These results suggest that the D3 receptor protein is expressed in rat brain.
Amino Acid Sequence↗
Development of polyclonal anti-D2 dopamine receptor antibodies using sequence-specific peptides.
Multiple subtypes of dopamine receptors with similar properties have been described. Ligands that have been shown to interact with a single subtype of receptor do not yet exist. The use of immunologic methods provides an alternative approach to distinguish receptors and receptor isoforms. Synthetic peptides corresponding to portions of the third intracellular loops of the two isoforms of the rat D2 dopamine receptor were used to elicit polyclonal antipeptide antibodies. Peptide D2-244 is unique to the D2L isoform, whereas peptide D2-284 is present in both the D2L and the D2S isoforms. Rabbits were immunized monthly with peptide coupled to keyhole limpet hemocyanin. The immunogenicity of the peptides was established using a solid-phase radioimmunoassay. Both immunogens elicited antipeptide antibodies within 10 weeks of the primary immunization, with titers of at least 1/10(4). An immunoprecipitation assay using receptors in digitonin-solubilized extracts of rat or canine caudate labeled with the high affinity D2 antagonist 125I-NCQ 298 showed that antipeptide antisera could recognize solubilized D2 receptors. At a dilution of 1/1000, antisera to peptide D2-284 quantitatively immunoprecipitated 125I-NCQ 298 binding sites from both rat and canine striatal tissue, whereas antisera against peptide D2-244 immunoprecipitated 40% of the D2 receptors solubilized from rat caudate. The selectivity of the antisera was determined using 293 cells transfected with cDNA encoding the D2L or the D2S isoform of receptor. Antisera to D2-284, at a dilution of 1/1000, were able to quantitatively immunoprecipitate receptor from both 293-D2L and 293-D2S cells. Antisera to D2-244 were specific for the D2L isoform, immunoprecipitating 125I-NCQ 298 binding sites from 293-D2L cells but not from 293-D2S cells. Anti-D2-284 specifically recognized multiple bands of 100 kDa, 68 kDa, and 50 kDa in immunoblots of denatured preparations of rat caudate. Immunohistochemical studies with anti-D2-284 demonstrated the presence of the D2 receptor in several regions of rat brain. Immunostaining was most dense in the striatum, with a lateral to medial gradient and patches of lighter staining. Immunoreactivity was negligible with preimmune serum or peptide-blocked immune serum. Immunoreactive processes were seen in the nucleus accumbens and ventral pallidum, as well as in the hypothalamus. The high affinity binding of agonist to D2 dopamine receptors was disrupted by anti-D2-284 but not anti-D2-244 antisera, implicating the internal region of the third intracellular loop represented by peptide D2-284 as a potential determinant of receptor-guanine nucleotide-binding protein coupling.
Amino Acid Sequence↗
Comparison of the expression, transcription and genomic organization of D2 dopamine receptors in outbred and inbred strains of rat.
Three outbred (Sprague-Dawley, Wistar and Long-Evans) and five inbred (Brown-Norway, Buffalo, DA, Fisher and Lewis) strains of rat were used to investigate the extent of genetic variation in the expression and organization of the rat D2 receptor locus. Radioligand binding studies were performed using 125I-iodobenzamide ([125I]IBZM), a high-affinity antagonist for D2 dopamine receptors, to determine the extent of variation in the expression of D2 receptors in these strains of rat. A comparison of the affinities (Kd = 0.26-0.38 nM) and densities (450-580 fmol/mg of protein) of binding sites for [125I]IBZM in the striatum of the eight strains of rat did not reveal statistically significant differences. Solution hybridization using 32P-labeled riboprobes complementary to the coding region of the third intracellular loop of the D2 receptor was used to investigate the extent of variation in transcription of the long (D2L) and short (D2S) isoforms of D2 receptor mRNA in rat striatal tissue. The level of expression of these two mRNA isoforms was found to be invariant in the strains of rats that were examined. The genomic organization of the D2 receptor locus for each strain of rat was compared using Southern blot hybridization. Southern blots were hybridized with a DNA probe that codes for the D2L receptor isoform. Restriction fragment lengths were conserved between each rat strain for genomic DNA digested with BamHI, EcoRI, HindIII, PstI and TaqI. Restriction fragment length polymorphisms (RFLPs) were identified when genomic DNA was digested with XbaI or MspI. The XbaI polymorphism was mapped to within 2 kb of the exon coding for the third intracellular loop of the D2 receptor. Both RFLPs differentiated Sprague-Dawley and Brown-Norway rats from Wistar, Long-Evans, Buffalo, DA, Lewis and Fisher rats. The RFLPs for the rat D2 receptor locus provide genetic markers that can be used with classic genetic studies to determine whether strain differences in behavior and/or in the response to pharmacologic intervention are determined by genetic elements linked to the D2 receptor locus.
Animals↗
Effect of N-alkylation on the affinities of analogues of spiperone for dopamine D2 and serotonin 5-HT2 receptors.
Two series of N-substituted spiperone analogues were prepared and evaluated in vitro to measure their affinities for dopamine D2 and serotonin 5-HT2 receptors. Substitution of the amide nitrogen with an alkyl group of five carbon units or less resulted in analogues displaying a low selectivity for D2 compared to 5-HT2 receptors. However, a moderate improvement in selectivity for D2 receptors was observed with N-benzylspiperone. Substitution at either the ortho or para position of the benzyl group resulted in a further reduction in affinity for 5-HT2 receptors and improvement in the selectivity ratio. Examination of N-substituted analogues of spiperone may provide insights into the topography of the antagonist binding region of the 5-HT2 receptor. The results also suggest that an 18F-labeled analogue of N-(4-nitrobenzyl)spiperone (4p) may be a suitable tracer for studying D2 receptors with positron emission tomography since this compound displays a high selectivity for D2 receptors relative to that of spiperone and N-methylspiperone.
Alkylation↗
Quantitation of isotypes of D2 receptors using solution hybridization.
Solution hybridization was used to quantitate the expression of the long and short splice variants of mRNA coding for the dopamine D2 receptor. Sequences corresponding to 450 bases of the coding region of the long form of the third intracellular loop and the full-length coding sequences of the long and short isoforms of the D2 receptor were amplified using PCR and cloned into a Bluescribe (pBS+) vector. Phage polymerases were used to synthesize riboprobes complementary to the third intracellular loop. Hybridization was carried out using sense strand RNA or total RNA isolated from tissue or cells. After hybridization and digestion with nucleases, the hybridization products were size-fractionated on a urea acrylamide gel. The RNA coding for the D2 receptor appeared as two distinct bands. One band (438 base pairs) represents the long form of the receptor and a second band (285 base pairs) represents the short form of the RNA coding for the receptor. Quantitation of the amount of RNA present suggests that in the striatum the long form of RNA coding for the receptor is expressed at higher levels than is the short form. In a cell line containing dopamine receptors derived from the pituitary (SUPI), the long form predominates to an even greater extent than in the striatum.
Amino Acid Sequence↗
Subtypes of serotonin receptors and anxiolytic drugs: model systems for the study of 5-HT receptors.
Explore the source record for details and available documents.
Animals↗
Down-regulation of beta-adrenergic receptors by pindolol in Gs alpha-transfected S49 cyc- murine lymphoma cells.
The role of the alpha subunit of the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase (GS alpha) in the down-regulation of beta-adrenergic receptors by pindolol was studied in S49 cyc- cells (normally GS alpha-deficient) transfected to express functional recombinant rat GS alpha. An inducible cell line (S49 GS alpha IND) was derived from S49 cyc- cells transfected with a vector containing the full-length coding sequence of GS alpha under the inducible control of the mouse mammary tumor virus long-terminal repeat promoter. GS alpha was not detectable in S49 GS alpha IND cells by immunoblot or by ADP-ribosylation in the presence of cholera toxin and [alpha-32P]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol-stimulated cyclic AMP accumulation increased within 3 h. After 15 h, GS alpha was present at a level 40-50% of that found in S49 wild-type (WT) cells as measured either by immunoblot analysis or by [alpha-32P]ADP-ribosylation. Membranes prepared from GS alpha IND cells grown in the presence of dexamethasone bound agonist with high affinity, and this binding was sensitive to guanine nucleotides. A second vector, DzbGS alpha +, contained the coding sequence of GS alpha under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc- cells, and the resulting cells, S49 GS alpha CST cells, expressed GS alpha at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol-stimulated cyclic AMP accumulation in S49 GS alpha CST cells was at least as great as in S49 WT cells. When cells were grown in the presence of dexamethasone, exposure to 50 nM pindolol for 12 h down-regulated the density of beta-adrenergic receptors in S49 WT cells to 60% of that in cells grown in the absence of pindolol, but pindolol had no effect on the density of receptors on cyc- or GS alpha IND cells. When GS alpha CST cells were exposed to 50 nM pindolol for 12 h, the density of beta-adrenergic receptors was down-regulated by the same amount as in S49 WT cells. These results suggest that GS alpha is necessary to restore the ability of pindolol to down-regulate beta-adrenergic receptors in S49 cyc- cells and that the protein must be expressed at a level comparable to that found in S49 WT cells.
Adenylyl Cyclases↗