Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Agonists for GTP-binding protein (G protein)-coupled receptors are thought to bind with high affinity to the complex of receptor and G protein. Nonhydrolyzable GTP analogs have been shown to disrupt this complex and reduce the binding affinity for many agonists. Antagonists are thought to bind to the receptor whether or not it is coupled to the G protein, and therefore binding remains unchanged in the presence of GTP analogs. The binding of the serotonin 5-hydroxytryptamine (5-HT)2 receptor agonists serotonin (5-HT) and 4-bromo-2,5-dimethoxyphenylisopropylamine is not affected by the presence of GTP analogs when the cloned 5-HT2 receptor is expressed in the 293 human embryonic kidney cell line. The same receptor expressed in mouse NIH3T3 cells is partially sensitive to GTP analogs. Both cell lines have similar proportions of agonist and antagonist binding sites, and agonist stimulation of both cell lines leads to a robust increase in phosphoinositide hydrolysis. Differences in GTP metabolism in 293 cells is not likely to be the cause of the observed difference in inhibition of agonist binding, because the cloned 5-HT1A serotonin receptor expressed in these cells is sensitive to GTP analogs. The GTP-insensitive agonist binding is best explained by the existence of a G protein-receptor complex in 293 cells that is not sensitive to GTP analogs. Such a G protein-receptor complex may explain the fraction of agonist binding in the brain that is not sensitive to GTP analogs.
Mol Pharmacol 1993 Jun
PMID:High affinity agonist binding to cloned 5-hydroxytryptamine2 receptors is not sensitive to GTP analogs. 831 23

Serotonin 5-hydroxytryptamine (5-HT)2 receptors are implicated in the etiology of mental disease and depression. Drugs that interact with the 5-HT2 receptor are used therapeutically to treat such illnesses, and their mechanisms of action are of great interest. In this study 5-HT2 receptor-ligand interactions were examined by site-directed mutagenesis in which three aspartic acid to asparagine mutants (Asn-120, Asn-155, and Asn-172) were created and expressed in NIH3T3 cells. The Asp-120 to asparagine mutant exhibited the same affinity for 125I-lysergic acid diethylamide (125I-LSD) as did the wild-type receptor and showed a decreased and GTP-insensitive binding affinity for the agonists 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (approximately 10-fold) and the antagonists ketanserin and mianserin (approximately 10-fold) but not spiperone. The mutation also abolished agonist-stimulated formation of [3H]polyphosphoinositides (PI). The Asn-155 mutant showed reduced binding affinity for 125I-LSD (Kd, 2.8 nM versus 0.6 nM for the wild-type receptor) and had reduced affinity for agonists (approximately 30-fold) and for antagonists (14-75-fold). However, the Asn-155 receptor retained GTP sensitivity and the ability to stimulate PI formation. The Asn-172 mutant retained the wild-type Kd value for 125I-LSD, exhibited only approximately 5-fold reduced affinity for 5-HT and (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane while retaining GTP-sensitive agonist binding showed no change in affinity for ketanserin, and had a small decrease in mianserin and spiperone binding (approximately 6-fold). The Asn-172 receptor also retained the ability to form PI. These results indicate that Asp-120 is necessary for allosteric activation of the guanine nucleotide-binding protein. Asp-155 is necessary for high affinity binding, probably by acting as a counterion for the amine group of the ligand. The different effects of the three mutations on ketanserin, mianserin, and spiperone binding affinity suggest that these antagonists may share overlapping but different binding domains. The information provided by this study may facilitate the design of therapeutic site-selective compound based on the structure of the 5-HT2 receptor.
Mol Pharmacol 1993 Jun
PMID:Site-directed mutagenesis of the serotonin 5-hydroxytrypamine2 receptor: identification of amino acids necessary for ligand binding and receptor activation. 831 24

Modulation of the inositol 1,4,5-trisphosphate (IP3)-mediated signal transduction pathway by the inhalational anesthetic enflurane was studied in Xenopus oocytes expressing mouse and human cortical mRNA. We found that enflurane significantly inhibited ion currents activated by m1 muscarinic and 5-hydroxytryptamine (5-HT)1c receptors. This inhibition was dependent upon the concentration of acetylcholine or 5-HT, with large inhibition (80-89%) of low concentrations and small inhibition (8-44%) of high concentrations of acetylcholine and 5-HT. Similar effects were found with either mouse or human receptors. To investigate the mechanism of enflurane action, ion currents induced by intracellular injection of guanosine 5'-(3-O-thio)triphosphate and IP3 were examined. Enflurane strongly suppressed the guanosine 5'-(3-O-thio)triphosphate-activated current but not the IP3-activated current. These results suggest that an inhalational anesthetic can disrupt the function of mouse and human brain phosphatidylinositol-linked receptors by selectively inhibiting the guanine nucleotide-binding protein activity.
Mol Pharmacol 1993 Jun
PMID:Enflurane inhibits the function of mouse and human brain phosphatidylinositol-linked acetylcholine and serotonin receptors expressed in Xenopus oocytes. 831 25

Serotonin (5-hydroxytryptamine; 5-HT) has recently been shown to induce collagenase production in myometrial smooth muscle cells (Jeffrey et al. (1991) J. Cell. Physiol. 146, 399-406) by activating transcription of the collagenase gene (Wilcox et al. (1992) J. Biol. Chem. 267, 20752-20757) following an interaction with the 5-HT2 receptor (Rydelek-Fitzgerald et al. (1993) Mol. Cell. Endocrinol. 91, 67-74). These studies were performed to investigate factors controlling the regulation of 5-HT2 receptors in these cells. Northern blot analysis indicates that serotonin increases levels of 5-HT2 receptor mRNA in cells by approximately 4-fold. Detectable increases in mRNA levels occur within 2 h after administration of serotonin with maximal levels occurring after 12 h. The 5-HT2 receptor antagonists, ketanserin and spiperone, inhibit the serotonin-mediated increase in receptor mRNA. Selective 5-HT2 receptor agonists ((+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI) and quipazine) mimic the effect of serotonin, whereas 5-HT1 and 5-HT3 receptor agonists ((+/-)-8-hydroxydipropylaminotetralin (8-OH-DPAT), 1-(3-chlorophenyl)piperazine dihydrochloride (mCPP), m-phenylbiguanide) have no effect. These data demonstrate that serotonin induces an increase in 5-HT2 receptor mRNA by interacting with the 5-HT2 receptor itself. Nuclear run-on analysis revealed that serotonin increases the initiation of 5-HT2 receptor mRNA synthesis. Moreover, the protein synthesis inhibitor, cycloheximide, prevents the induction of the mRNA for the receptor, demonstrating that serotonin-dependent increases in 5-HT2 receptor transcription require de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1993 Apr
PMID:Serotonin-mediated 5-HT2 receptor gene regulation in rat myometrial smooth muscle cells. 831 29

Rat cerebellar granule cells express 5-hydroxytryptamine (5-HT)2 receptors that mediate phosphoinositide turnover by a pertussis toxin-sensitive mechanism. Prestimulation of these neurons with 10 microM 5-HT or (+/-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane [(+/-)-DOI], a putative 5-HT2 receptor agonist, resulted in a time-dependent desensitization of the phosphoinositide response to 5-HT. The desensitization was detected within 30 min after prestimulation and reached a maximum (about 80%) decrement at 8 hr. However, [3H]ketanserin binding to 5-HT2 receptors in crude membranes or intact cerebellar granule cells was increased by treatment with 5-HT or DOI, in a time- and concentration-dependent manner. The increase occurred after the onset of desensitization and was fully manifest (about 160-190%) at 4 hr after stimulation. Although the Bmax and Kd were unchanged at 1 hr after 5-HT or DOI treatment, both parameters were significantly increased at 4 and 24 hr. The amount of 5-HT2 receptor mRNA detected by Northern blot hybridization using a 5-HT2 receptor-specific riboprobe was increased in parallel with the up-regulation of 5-HT2 receptor binding sites. Thus, an increase in 5-HT2 receptor mRNA was detected within 2 hr after 5-HT or DOI prestimulation, reached a maximum around 4 hr, and remained at a plateau for at least 24 hr. The levels of total RNA, m3 muscarinic acetylcholine receptor mRNA, and beta-actin mRNA were not significantly affected by these treatments. Our results demonstrated that 5-HT2 receptor binding sites and their mRNA undergo a paradoxical induction during persistent agonist stimulation.
Mol Pharmacol 1993 Mar
PMID:Paradoxical increase of 5-hydroxytryptamine2 receptors and 5-hydroxytryptamine2 receptor mRNA in cerebellar granule cells after persistent 5-hydroxytryptamine2 receptor stimulation. 838

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.
Mol Pharmacol 1993 May
PMID:Effects of agonists, partial agonists, and antagonists on the regulation of 5-hydroxytryptamine2 receptors in P11 cells. 838 88

The molecular processes by which agonists and antagonists bind to serotonin2 [5-hydroxytryptamine (5-HT2)] receptors are currently unknown. Three molecular models have proposed that conserved aromatic residues help to anchor the phenyl ring of 5-HT via stacking or pi-pi-type interactions with the 5-HT2 receptor. To test these models we made single point mutations (Phe339-->Leu339 and Phe340-->Leu340) of two aromatic residues that are conserved among all guanine nucleotide-binding protein-coupled 5-HT receptors and a single point mutation (Phe125-->Leu125) that exchanges a 5-HT2 for a 5-HT1c sequence. [3H]Mesulergine binding was abolished by Phe340-->Leu340 and unchanged with the Phe339-->Leu339 and Phe125-->Leu125 mutations, whereas [3H]ketanserin binding affinity was diminished by the Phe339-->Leu339 mutation and unchanged by Phe340-->Leu340 and Phe125-->Leu125. We also found that the affinities of three ergot derivatives (mesulergine, methysergide, and lisuride) were decreased by 88-1079-fold with only the Phe340-->Leu340 mutation. We also discovered that 4-[125I]iodo-2,5-(dimethoxy)phenylisopropylamine (DOI) binding was abolished in COS-7 cells expressing 5-HT2 (Phe340-->Leu340) receptors but maintained in cells expressing the Phe339-->Leu339 and Phe125-->Leu125 mutations. Additionally, the Ki values for several agonists and partial agonists (5-HT, DOI, m-chlorophenylpiperazine, trifluoromethylphenylpiperazine, bufotenine, and MK-212) were greatly diminished (26-14,000-fold decrease) only with the Phe340-->Leu340 receptor mutation. Finally, the Phe340-->Leu340 mutant receptors displayed an attenuated or abolished ability to augment phosphoinositide hydrolysis in COS-7 cells with four separate agonists (5-HT, MK-212, bufotenine, and quipazine). Taken together, these results are consistent with the idea that agonists and certain ergot derivatives anchor to 5-HT2 receptors, in part, via specific interactions with aromatic residue Phe340 located in transmembrane region VI.
Mol Pharmacol 1993 May
PMID:A single point mutation (Phe340-->Leu340) of a conserved phenylalanine abolishes 4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine and [3H]mesulergine but not [3H]ketanserin binding to 5-hydroxytryptamine2 receptors. 838 89

Inhibitory (glycine and gamma-aminobutyric acid type A) and excitatory (nicotinic acetylcholine and serotonin 5-hydroxytryptamine type 3) receptors of the ligand-gated ion channel superfamily are related by both structural similarities and primary sequence identity. One invariant feature of all members of this receptor superfamily is the presence of an extracellular disulfide loop motif. This structural motif has been modeled, and Cockcroft et al. [Proteins 8:386-397 (1990)] have suggested that it forms the agonist binding site of the ligand-gated ion channel receptors. Using site-directed mutagenesis of the inhibitory glycine receptor (GlyR), we have specifically tested this hypothesis. The lysine residue at position 143 is proposed to form the binding site for the negatively charged carboxyl group of the agonist glycine. Differing residues at this position in other ligand-gated receptors are proposed to confer agonist specificity. The aspartic acid residue at position 148 is an invariant residue in every known subunit of the ligand-gated ion channel receptor superfamily. This residue has been proposed as the binding site for the positively charged amino group of the various agonists. Mutation of the lysine at position 143 to alanine resulted in essentially unaltered GlyRs, showing only modest decreases in strychnine affinity (Kd, 8.1 +/- 1.4 nM versus 13.4 +/- 1.3 nM), glycine displacement of strychnine binding (Ki, 25 +/- 5 microM versus 49 +/- 9 microM), and glycine activation of chloride currents (EC50, 27 +/- 6 microM versus 114 +/- 14 microM). Thus, we conclude that Lys-143 does not play a major role in either agonist or antagonist binding or agonist activation of the GlyR. Mutation of Asp-148 to either alanine or asparagine disrupted the expression and/or assembly of the receptor, and no binding sites or ion channels were expressed on the cell surface. The conservative mutation of the aspartic acid at position 148 to glutamic acid (D148E) allowed the expression of receptors, although with reduced efficiency. The D148E GlyRs showed a 1 order of magnitude decrease in strychnine affinity (Kd, 8.1 +/- 1.4 nM versus 82 +/- 21 nM), without any change in the glycine displacement of strychnine binding (Ki, 25 +/- 5 microM versus 29 +/- 8 microM) or glycine activation of chloride currents (EC50, 27 +/- 6 microM versus 20 +/- 1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1993 Jul
PMID:The extracellular disulfide loop motif of the inhibitory glycine receptor does not form the agonist binding site. 839 21

Serotonin modulates a wide range of physiological functions by activating multiple receptors, which are coupled to various effector systems. Using a strategy based on amino acid sequence homology between 5-hydroxytryptamine (5-HT) receptors, we have isolated from a mouse brain library a cDNA encoding a new 5-HT receptor, 5-HTx, that activates adenylate cyclase. Amino acid sequence comparisons revealed that the 5-HTx receptor was a distant relative of previously cloned 5-HT receptors, with the highest percentage of homology (42%) being with the 5-HTdro1 receptor, a Drosophila 5-HT receptor positively coupled to adenylate cyclase. In COS-7 cells transiently expressing the 5-HTx receptor, 5-HT induced an increase in cAMP levels that was dose dependent and saturable (EC50 = 45 nM). Agonists displayed the following rank order of potencies: 5-carboxamidotryptamine > 5-methoxytryptamine > 5-HT > RU 24969 > 8-hydroxy-2-(di-n-propylamino)tetralin. The most efficient antagonists in inhibiting the stimulatory effect of 5-HT were methysergide, methiothepin, mesulergine, metergoline, clozapine, ergotamine, and (+)-butaclamol. Membranes of COS-7 cells expressing the 5-HTx receptor displayed a single saturable binding site for [3H]5-HT. The order of potencies of various drugs in displacing [3H]5-HT binding was similar to the order obtained in cAMP experiments. The pharmacological profile of this receptor does not correspond to the profile of any of the classic 5-HT receptor subtypes. Expression of 5-HTx mRNA was highest in brainstem and lower in forebrain, cerebellum, intestine, and heart. The 5-HTx receptor might therefore correspond to 5-HT1-like receptors that have been shown to induce relaxation in porcine vena cava and guinea pig ileum as well as tachycardia in cat heart. The high affinity of the 5-HTx receptor for neuroleptic agents such as (+)-butaclamol and clozapine suggests also that this receptor might play a role in certain neuropsychiatric disorders.
Mol Pharmacol 1993 Aug
PMID:Molecular cloning of a mammalian serotonin receptor that activates adenylate cyclase. 839 87

Serotonin [5-hydroxytryptamine (5-HT)] has been implicated in the pathophysiology of migraine, and the clinical efficacy of the 5-HT1B/5-HT1D receptor agonist sumatriptan points to neural and/or vascular 5-HT1D receptors as relevant targets in migraine therapy. We characterized the human and/or bovine 5-HT1D receptor subtype in cerebral blood vessels pharmacologically by correlation analysis and molecularly by Northern blot hybridization of cerebrovascular RNA extracts. Pharmacological analysis showed that sumatriptan was less potent than 5-HT in inducing contraction in freshly isolated human cerebral arteries and revealed an overall pharmacological profile positively and significantly correlated with that published for the 5-HT1D alpha (r = 0.746, p = 0.021) and 5-HT1D beta (r = 0.942, p = 0.0001) cloned human receptor subtypes. These results are suggestive of a contractile 5-HT1D beta receptor subtype but are not conclusive. However, Northern blots revealed the presence of mRNA transcripts for the 5-HT1D beta subtype, but not the 5-HT1D alpha subtype, in bovine (approximately 2.2 kilobases) and human (approximately 4.5 kilobases) cerebral blood vessels. Expression of either subtype could not be detected in intraparenchymal microvessels or capillaries isolated from bovine or human cerebral cortex. These results clearly indicate that the beneficial effect of sumatriptan in migraine attack, if vascularly related, is mediated by contractile 5-HT1D beta receptors most likely located on cerebral blood vessels at the surface of the brain. This study points to the 5-HT1D beta receptor subtype as the putative cerebrovascular target for migraine therapeutic agents.
Mol Pharmacol 1993 Aug
PMID:Expression of mRNA for the serotonin 5-hydroxytryptamine1D beta receptor subtype in human and bovine cerebral arteries. 839 88


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