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Query: UNIPROT:P06889 (Mol)
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This review will focus on the main lines of evidence that suggest the existence of multiple types of dopamine D-2 receptors. Dopamine D-2 receptors share structural elements suggesting that they belong to a gene superfamily classified as G-protein-coupled receptors and show an archetypical topology predicted to consist of seven putative transmembrane domains. Activation of D-2 receptors results in a variety of responses, including inhibition of cyclic AMP formation, inhibition of phosphoinositol turnover, increase of K-channel activity, and inhibition of Ca influx. The G protein(s) linking the D-2 receptors to these responses have not been completely identified, nor has the possible hierarchy of these regulatory proteins in transforming the incoming signal into a change of second-messenger levels. A lot of experimental data support the hypothesis that there are multiple signal-processing pathways activated by dopamine through D-2-receptor stimulation. Recently, the identification of dopaminergic drugs that discriminate among the different transduction pathways and the isolation of distinct cDNAs encoding proteins that share binding profile indicative of D-2 receptors clearly indicate multiple forms of D-2 receptors. Pharmacologically, at least two distinct categories of dopamine D-2 receptors exist in rat pituitary. The first (D-2a) is insensitive to BHT 920 and coupled to inhibition of adenylyl cyclase activity; the second (D-2b) is activated by BHT 920 and linked to voltage-dependent K channels. The two types of dopamine D-2 receptors differ in their structure, G-protein-coupled and effector. Each of the three basic receptor units shows a certain degree of heterogeneity, which may affect the quality and the kinetic of the response. This variety may represent the molecular basis for the diversity in pharmacological and functional profiles of different dopamine D-2 receptors located in various brain areas and peripheral tissues.
Mol Neurobiol
PMID:Pharmacological and molecular basis for dopamine D-2 receptor diversity. 213 94

Human genomic DNA and the HSV tk gene were cotransfected into mouse Ltk- cells and assayed for the acquisition of a Gs-coupled receptor to obtain cell lines expressing human receptors that are so far unavailable. The transfected cells were distributed into 96-well microtitration plates at a density such that after HAT (100 microM hypoxanthine, 1 microM aminopterin, and 10 microM thymidine) selection each well contained, on the average, two to three tk+ cell clones. After replication, half of them were tested for expression of a new phenotype: an adenylyl cyclase stimulatory receptor not normally expressed in the Ltk- recipient cell. The screen yielded a positive result on testing cells arising from the third transfection, the newly expressed receptor is that for arginine vasopressin, commonly referred to as type 2 or V2. DNA from primary transformants (HTB-1 cells) served to obtain secondary transformants by the same technique (HTB-2 cells). Pharmacological properties confirmed that this new receptor, which stimulates adenylyl cyclase activity 7- to 10-fold, is the human V2 receptor and not the activated homologous murine gene. The new cell line provides a permanent accessible source to study the human receptor, by-passing the need for human kidneys. The V2 receptor was susceptible to homologous down-regulation in the HTB-2 cell, but no down-regulation of the cell authentic prostaglandin E1 receptor was observed. The vasopressin receptor did not modify phospholipase-C activity in these cells as expected from V2 receptors. Thus, we successfully applied genomic DNA-mediated gene transfer and were able to develop a cell line expressing a Gs-coupled human receptor of low abundance and poor accessibility.
Mol Endocrinol 1990 Feb
PMID:Development and characterization of a mouse cell line expressing the human V2 vasopressin receptor gene. 213 94

The molecular basis for the effects of 4 beta-phorbol 12-myristate 13-acetate (PMA) on adenylyl cyclase activation was examined using site-directed mutants of the hamster beta-adrenergic receptor (beta AR) expressed in L cells. Phorbol ester activation of protein kinase C (PKC) in L cells transfected with wild-type beta AR caused at least three discernible effects on adenylyl cyclase activity, each with an EC50 of 20 to 50 nM, (i) a 2-3-fold increase in the Kact for epinephrine stimulation, (ii) a 2-3-fold increase in the maximal level (Vmax) of hormonal stimulation, and (iii) a decrease in the Gi-mediated inhibition of forskolin stimulation. Deletion from the beta AR of amino acid residues 259-262, which removes one of the two consensus sites for phosphorylation by PKC, eliminated (greater than 90%) the PMA-induced increase in the Kact, whereas the PMA-induced increase in the Vmax and loss of Gi-mediated inhibition were not affected by the deletion. Neither deletion of the other PKC consensus site in the beta AR (residues 343-348) nor truncation of the Ser/Thr-rich C-terminal domain (residues 354-418) affected the PMA-induced changes in adenylyl cyclase. The effects of PMA on Gi-mediated inhibition and the Vmax closely mimicked the action of islet-activating protein, consistent with a direct effect of PMA-activated PKC on Gi. In contrast, the effects on the Kact appear to be receptor specific. These results demonstrate that the consensus site for phosphorylation by PKC, found in the third intracellular loop of the beta AR, is required for the PMA-induced increase in the Kact for epinephrine stimulation. Use of L cells transfected with D(259-262)beta AR allowed the characterization of the postreceptor effects of PMA without interference from receptor-level effects.
Mol Pharmacol 1990 Sep
PMID:Identification of a specific domain in the beta-adrenergic receptor required for phorbol ester-induced inhibition of catecholamine-stimulated adenylyl cyclase. 216 78

Tissue levels of 3'-AMP were measured in several rat tissues and the sensitivities of the respective adenylyl cyclases were compared with respect to "P" site-mediated inhibition by 3'-AMP2'-deoxy-3'AMP (2'd3'-AMP), and 2',5'-dideoxyadenosine. IC50 values for these P site inhibitors of adenylyl cyclases varied widely among tissues, e.g., with skeletal muscle being least sensitive to 3'-AMP (IC50 greater than 170 microM) and brain being most sensitive (IC50 approximately 10 microM). These differences were noted when activation was with Mn2+ but diminished with Mn2+ plus forskolin and conceivably may reflect the distribution of different isozymes of adenylyl cyclase. 3'-AMP levels also varied significantly among rat tissues, with spleen having the highest levels (approximately 280 nmol/g), kidney, liver, heart, and brain having decreasing 3'-AMP content, and skeletal muscle levels being immeasureably low (less than 0.1 nmol/g). When rats were made diabetic with streptozotocin, the 3'-AMP content of livers increased from approximately 47 nmol/g in control animals to approximately 84 nmol/g, a change largely reversed by maintenance of diabetic animals with insulin. The data suggest that tissue 3'-AMP levels may be regulated and in certain tissues may be sufficient to inhibit adenylyl cyclase in vivo. Three potential sources of 3'-AMP and 2'd3'-AMP, the most potent naturally occurring P site inhibitors of adenylyl cyclase, were examined. No evidence was found for the formation of either nucleotide from the respective cyclic nucleotide by a unique cyclic nucleotide phosphodiesterase or from the respective nucleoside by a hypothetical adenosine 3'-kinase and ATP. Substantial 3'-AMP and 2'd3-AMP were formed by spleen and liver homogenates from the respective oligonucleotides (RNA, mRNA, and DNA) in a time- and protein-dependent manner. The data imply the existence of enzymes in these tissues to catalyze the formation of 3'-AMP and 2'd3'-AMP from nucleic acids and suggest that these activities may account for the formation of P site agonists under in vivo conditions. The data suggest that these P site inhibitors are a potential link between fluctuations in nucleic acid metabolism and altered sensitivity of membrane-bound adenylyl cyclase to stimulatory signals.
Mol Pharmacol 1990 Dec
PMID:Tissue levels, source, and regulation of 3'-AMP: an intracellular inhibitor of adenylyl cyclases. 217 5

Chronic exposure to ethanol results in heterologous desensitization of receptors coupled to adenylyl cyclase via Gs, the stimulatory guanine nucleotide regulatory protein. Ethanol-induced accumulation of extracellular adenosine is required for the development of heterologous desensitization (Nagy, L. E., Diamond, I., Collier, K., Lopez, L., Ullman, B., and Gordon, A. S., Mol. Pharmacol., in press). To understand the mechanism underlying ethanol-induced increases in extracellular adenosine, we examined the interaction of ethanol with the adenosine transport system in S49 lymphoma cells. We found that ethanol inhibited nucleoside uptake without affecting deoxyglucose or isoleucine transport. Inhibition of adenosine uptake was due to decreased influx via the nucleoside transporter. Thus, ethanol-induced increases in extracellular adenosine appear to be due to inhibition of adenosine influx. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol, i.e. ethanol no longer inhibited uptake. Consequently, ethanol no longer increased extracellular adenosine concentrations. Taken together with our previous studies, these results suggest that ethanol inhibition of adenosine influx leads to an increase in extracellular adenosine which causes an initial increase in intracellular cAMP levels and subsequent development of heterologous desensitization of cAMP signal transduction.
 ...
PMID:Ethanol increases extracellular adenosine by inhibiting adenosine uptake via the nucleoside transporter. 229 33

We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.
Mol Cell Biol 1988 May
PMID:Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. 245 17

Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1987 Apr
PMID:Reconstitution of somatostatin and muscarinic receptor mediated stimulation of K+ channels by isolated GK protein in clonal rat anterior pituitary cell membranes. 245 51

Distinct membrane receptors that elicit similar cellular responses may share elements of signal transduction. In the present study, rat hippocampal adenosine (AD) and 5-hydroxytryptamine (5-HT) receptors were chosen to test this possibility using biochemical and electrophysiological techniques. Responses elicited by the AD receptor that mediates the inhibition of forskolin-stimulated adenylyl cyclase activity in rat hippocampal membranes and hyperpolarization of resting membrane potential (RMP) in rat hippocampal pyramidal cells were characterized and compared, in the same preparation, with those analogous responses elicited by the 5-HT1A receptor. A series of AD agonists including the selective AD A1 agonist (R)-phenylisopropyladenosine [(R)-PIA] inhibited forskolin-stimulated adenylyl cyclase activity in rat hippocampal membranes in a concentration-dependent manner. Cyclopentyltheophylline (CPT), a selective AD A1 antagonist, was a potent, competitive antagonist of this response with a dissociation constant (Kb) of 6 nM (Schild analysis). The rank order of agonist EC50 values and antagonist Kb values, as well as stereoselectivity, are consistent with the classification of this receptor as the AD A1 receptor. Spiperone, a potent 5-HT1A antagonist, competitively antagonized 5-HT-mediated inhibition of forskolin-stimulated adenylyl cyclase activity in rat hippocampal membranes with a Kb value of 14 nM. Intracellular recording techniques revealed that AD, (R)-PIA, 5-HT, and 5-carboxyamidotryptamine (5-CT) elicited concentration-dependent hyperpolarization of RMP within the same hippocampal pyramidal cell. The maximal hyperpolarization obtained for the AD or 5-HT analogs was the same for individual pyramidal cells. CPT and spiperone antagonized the hyperpolarization by (R)-PIA and 5-CT, respectively. Saturating concentrations of spiperone failed to antagonize (R)-PIA-mediated responses and CPT did not block responses elicited by 5-HT in either the biochemical or electrophysiological preparations. The combination of saturating concentrations of 5-HT and (R)-PIA evoked nonadditive biochemical responses relative to those observed with (R)-PIA alone. Similarly, electrophysiological experiments conducted under voltage-clamp conditions demonstrated that maximally effective concentrations of AD and 5-CT exhibited nonadditive behavior. Because the amount of outward current elicited when these agonists were coperfused was significantly less than the algebraic sum of the currents evoked individually by these agents, we infer that a population of AD A1 and 5-HT1A receptors activates a common pool of guanine nucleotide-binding proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1989 Apr
PMID:Pertussis toxin-sensitive guanine nucleotide-binding protein(S) couple adenosine A1 and 5-hydroxytryptamine1A receptors to the same effector systems in rat hippocampus: biochemical and electrophysiological studies. 249 34

The developing chick heart was employed as a model system to explore temporal correlations between the onset of pharmacological sensitivity to adenosine analogs and the appearance of A1 adenosine receptors coupled to adenylyl cyclase. A characterization of the developmental profile for adenosine analog-induced negative chronotropic response revealed that isolated atria from 5- and 6-day embryos were unresponsive to adenosine analogs. The onset of pharmacological sensitivity occurred on embryonic day 7, as evidenced by a 27% reduction in atrial beating rate in the presence of 2-chloradenosine (2-CIA) (30 microM). The sensitivity of embryonic atria to 2-CIA increased continuously from day 7 to day 12 in ovo, when the atria became fully responsive to the negative chronotropic effect of this adenosine analog. In order to evaluate whether the developmental increase in pharmacological sensitivity to 2-CIA reflected changes in the number of A1 adenosine receptors, the ontogenesis of A1 adenosine receptors was assessed using the antagonist radioligand 8-cyclopentyl-1,3-[3H]dipropylxanthine as a probe. Cardiac membranes from day 5 and day 6 embryos possessed approximately one third of the maximum number of A1 adenosine receptors expressed at later embryonic ages. Additionally, agonist/[3H] DPCPX competition curves revealed that the high affinity state receptors comprised a larger proportion of the total receptor population in membranes from day 6 as compared with day 12 embryos. These results suggest that there are pharmacologically inactive A1 receptors in hearts from day 5 and day 6 embryos. The developmental change in A1 receptor-mediated negative chronotropic response paralleled the increase in [3H]DPCPX binding sites from embryonic day 7 to day 10. Thus, a large fractional occupancy of A1 adenosine receptors is required to express negative chronotropy during this period of embryonic development. Studies of the sensitivity of adenylyl cyclase to inhibition by cyclopentyladenosine as a function of ontogenesis revealed that cyclopentyladenosine inhibited basal adenylyl cyclase activity to a similar maximal extent from embryonic day 5 through day 16. The efficacy of cyclopentyladenosine as an inhibitor of adenylyl cyclase activity was, therefore, stable during a developmental period when A1 receptor density increased approximately 3-fold. Hence, only a fraction of the A1 receptors present during embryogenesis need to be coupled to produce a maximum response with respect to adenylyl cyclase inhibition, which is an indication of the presence of spare receptors. receptors.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1989 May
PMID:Development of pharmacological sensitivity to adenosine analogs in embryonic chick heart: role of A1 adenosine receptors and adenylyl cyclase inhibition. 249 36

In intact membranes as well as after reconstitution into phospholipid vesicles, pertussis toxin (PT)-mediated ADP-ribosylation of G proteins causes loss of receptor-mediated regulation of effectors and/or G protein-mediated regulation of receptor binding. Studies were carried out to test which of several discrete steps known to constitute the basal and receptor-stimulated regulatory cycles of Gi proteins are affected by PT. Experiments with the Gs-deficient Gi-regulated adenylyl cyclase of cyc- S49 cell membranes indicated that PT blocks Gi activation by GTP without affecting GDP dissociation or GTP binding to a major extent. This suggested that the block lies in the transition of inactive GTP-Gi to active GTP-Gi (G to G* transition). Experiments with purified Gi in solution and after incorporation into phospholipid vesicles showed that PT does not increase or decrease the intrinsic GTPase activity of Gi. Experiments in which Gi was incorporated into phospholipid vesicles with rhodopsin, a receptor that interacts with Gi to stimulate the rate of guanosine 5'-O-(3-thio)triphosphate binding and GTP hydrolysis, indicated that PT does not affect the basal GTPase activity of Gi, but blocks its activation by the photoreceptor. Taken together the results indicate that PT-mediated ADP ribosylation has two separate effects, one to block the interaction of receptor with Gi and another to impede the GTP-induced activation reaction from occurring, or that PT has only one effect, that of blocking interaction with receptors. In this latter case the present results add to a mounting series of data that are consistent with the hypothesis that unoccupied receptors are not inactive, but exhibit a basal agonist-independent activity responsible for the various effects of GTP observed on G protein-coupled effector functions in intact membranes.
Mol Endocrinol 1989 Jul
PMID:Studies on nucleotide and receptor regulation of Gi proteins: effects of pertussis toxin. 250 4


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