Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A permanently transformed cell line derived from human embryo renal cortical cells (HEK293) has been investigated for the retention of renal-specific properties. The cell line is epithelioid in growth on plastic, and transmission electron microscopy demonstrates the formation of apical zonae occludentes. There is no prominent brush-border. The response of HEK293 cell adenylate cyclase is noteworthy for the response to vasoactive intestinal peptide (half-maximal activation at 0.9 nM). The HEK adenylate cyclase response to VIP is specific, with related peptides such as glucagon and secretin being ineffective. The response to VIP is competitively antagonized by the VIP receptor antagonist (4Cl-D-Phe6,Leu17)-VIP.
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PMID:A cultured human renal epithelioid cell line responsive to vasoactive intestinal peptide. 216 76

A series of mutant porcine calcitonin receptors with progressively truncated carboxy termini have been expressed in COS and HEK 293 cells. All forms of the receptor, including those totally lacking the cytoplasmic tail, were able to bind 125I-labeled salmon calcitonin. However, removal of C-terminal domains resulted in multiple functional changes in the receptor. First, compared with the wild type receptor, affinity of binding of salmon calcitonin was increased for truncated receptors, whether determined in intact transfected cells or in cell membranes. Second, internalization of the ligand-receptor complex was greatly attenuated for mutants truncated by 44 or 83 amino acids but not for an intermediate form truncated by 63 amino acids. Third, truncation affected signal transduction, which for the porcine calcitonin receptor occurs by generation of intracellular cAMP and Ca2+. The magnitude of adenylate cyclase responses was much reduced for the same mutants defective in internalization. Under conditions where expression of each receptor form was approximately equal, the magnitude of intracellular Ca2+ responses was decreased by C-terminal truncation. These results draw attention to the functional significance of the cytoplasmic tail of the porcine calcitonin receptor and suggest intramolecular interactions between the carboxy terminus and other receptor domains and/or cellular regulatory elements.
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PMID:Truncation of the porcine calcitonin receptor cytoplasmic tail inhibits internalization and signal transduction but increases receptor affinity. 770 57

Somatostatin (SRIF) SS-2 binding sites were originally defined in rat brain cerebral cortex membranes using [125I]Tyr11-SRIF-14 in the presence of 120 mM NaCl. These sites were characterized by their high affinity for SRIF-14 and SRIF-28, but very low affinity for cyclic peptides such as octreotide (SMS 201-995) and seglitide (MK 678). The characteristics of SS-2 sites are reminiscent of 125I]CGP 23996-labelled sites in rat brain which have been termed SRIF-2 sites. In the present study, the pharmacological profile of SS-2 sites was determined in radioligand binding studies performed in rat cortex membranes using [125I]SRIF-14 in the presence of 120 mM NaCl and compared to that of human SSTR-1 receptors expressed in human embryonic kidney (HEK 293) cells, using [125I]SRIF-14. The rank orders of affinity of a variety of SRIF analogues and synthetic peptides for SS-2 binding sites and recombinant human SSTR-1 receptors were very similar and correlated highly significantly (r = 0.99). However, SS-2 binding correlated also with binding to recombinant SSTR-4 receptors (r = 0.91). Autoradiographic studies were performed using the radioligand [125I]CGP 23996 which has been claimed to label selectively SRIF-2 binding sites and compared with the distribution of SSTR-1 receptor mRNA determined using in situ hybridization in rat brain. Although some overlap was observed between the distribution of SSTR-1 mRNA and [125I]CGP 23996 binding sites, the latter were clearly more widespread, suggesting this ligand to label SSTR-1 and other sites. In addition, inhibition of forskolin-stimulated adenylate cyclase was investigated in HEK 293 cells transfected with human SSTR-1 receptors; a variety of SRIF analogues and short synthetic peptides behaved as agonists at adenylate cyclase and displayed a rank order of potency highly similar to that observed for these compounds at SS-2 binding sites. Seglitide acted as an antagonist at SSTR-1 receptor mediated inhibition of adenylate cyclase activity with a pKB of 4.42. It is concluded that the pharmacological profile of SS-2 binding sites resembles most closely that of SSTR-1 receptors (although similarities with SSTR-4 receptors were observed), that [125I]CGP 23996 labels presumably several SRIF receptors in rat brain, and that SSTR-1 receptors are negatively and efficiently coupled to adenylate cyclase activity.
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PMID:Pharmacological identity between somatostatin SS-2 binding sites and SSTR-1 receptors. 778 6

Two rat calcitonin (CT) receptor isoforms; C1a and C1b, are identical except for the presence of a 37-amino acid insert in the second extracellular domain of C1b. The functional consequences of this insert were examined after stable expression of these receptors into HEK-293 cells. In binding competition studies, dissociation of [125I]salmon CT ([125I]sCT) from C1b cells was rapid and complete, in contrast to dissociation from C1a cells, which was slow and incomplete, as seen with other CT receptor preparations. In these studies, C1a receptors displayed high affinity for salmon CT (Kd, 0.5 +/- 1.3 nM) and a slightly lower affinity for pig CT. Human CT competed more weakly for binding of [125I]CT. Although the relative affinities of the ligands were maintained for C1b receptors, the affinity for sCT was lower (Kd, 23 +/- 2 nM) and pig CT was approximately 10-fold less potent than sCT. Human and rat CT failed to compete with [125I]sCT even at 1 microM with the C1b receptor. Both receptors influence multiple effector systems, indicating coupling to multiple G-proteins. The CT peptides activated adenylate cyclase with relative efficacies consistent with the binding competition potencies. In addition, both receptor isoforms mediated a rapid increase in the levels of intracellular calcium after a CT challenge. These results show that an extracellular modification in the rat CT receptor results in altered ligand recognition as well as altered binding kinetics, but does not modify their ability to generate multiple second messengers.
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PMID:Isoforms of the rat calcitonin receptor: consequences for ligand binding and signal transduction. 801 52

We have utilized the polymerase chain reaction technique to selectively amplify a G protein-coupled receptor cDNA from rat kidney proximal convoluted tubule mRNA, which exhibits high homology with previously cloned serotonin receptors. Sequencing of a full-length clone isolated from a rat hippocampal cDNA library revealed an open reading frame of 1,212 base pairs encoding a 404-residue protein with seven hydrophobic regions predicted to represent transmembrane-spanning domains. Within the transmembrane regions, this receptor was found to be 44-50% identical with various members of the 5-HT1, 5-HT5, and 5-HT6 subfamilies with lower (37-40%) homology to the 5-HT2-like receptors. Northern blots revealed a approximately 3.6-kilobase transcript localized in various brain regions with the following rank order of abundance: hypothalamus > hippocampus = mesencephalon > cerebral cortex = olfactory bulb > olfactory tubercle. Expression of this clone in COS-7 cells resulted in the appearance of high affinity, saturable binding of [3H]lysergic acid diethylamide ([3H]LSD; KD = 5 nM) and [3H]serotonin ([3H]5-HT; KD = 1 nM). Among endogenous biogenic amines, only 5-HT completely inhibited radioligand binding. The inhibition of radioligand binding by other serotonergic agents revealed a pharmacological profile that does not correlate with any previously described serotonin receptor subtype. In addition, this receptor exhibits high affinity for a number of tricyclic antipsychotic and antidepressant drugs including clozapine, loxapine, and amitriptyline. In HEK-293 cells stably transfected with this receptor, serotonin elicits a potent stimulation of adenylylcyclase activity. The distinct structural and pharmacological properties of this receptor suggests that it represents a completely novel serotonin receptor subtype, which we propose to designate 5-HT7. Based on its pharmacology and its localization to limbic and cortical regions of the brain, it is likely that this receptor may play a role in several neuropsychiatric disorders that involve serotonergic systems.
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PMID:Molecular cloning and expression of a 5-hydroxytryptamine7 serotonin receptor subtype. 839 62

Molecular cloning revealed the existence of at least five pharmacologically different dopamine receptors in vertebrates. Functionally, dopamine receptors either activate (D1-type), inhibit or do not interact with adenylate cyclase (D2-type). A recently cloned dopamine receptor from Drosophila melanogaster shares many structural and functional properties with vertebrate D1-type receptors but the pharmacological properties are very different. In contrast to most aminergic receptors, DmDop1 contains a long N-terminal extension. Here we describe a deletion-mutagenesis approach to study whether the N-terminus of DmDop1 participates in receptor-ligand interactions. All mutants gave rise to functional receptors after heterologous expression in HEK 293 cells. The pharmacological properties, however, remained unchanged. A comparison of DNA and deduced amino acid sequences revealed that some Drosophila strains express a truncated version of the DmDop1 receptor.
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PMID:Functional properties of Drosophila dopamine D1-receptors are not altered by the size of the N-terminus. 863 55

We have studied the functional properties of an alternately spliced form of sheep testicular FSH receptor cDNA that codes for a protein similar to a previously described active receptor but differs in the carboxy terminus in sequence and is also shorter by 25 residues. The receptor expressed in HEK 293 cells fails to activate adenylate cyclase. Cotransfection of stably expressing cells bearing FSH receptor that activates (Gs) adenylate cyclase with the altered receptor cDNA abrogates hormone response. In cells expressing this cDNA. FSH also inhibited cyclic AMP accumulation induced by non hormonal agents such as forskolin and cholera toxin which bypass the receptor. We propose that this altered receptor is a dominant negative receptor which may be coupled to G1 protein(s) or other inhibitory mechanisms.
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PMID:Follitropin signal transduction: alternative splicing of the FSH receptor gene produces a dominant negative form of receptor which inhibits hormone action. 883 80

UMR106-06 cells predominantly express the C1a isoform of the rat calcitonin (CT) receptor (CTR). We have compared the homologous regulation of the C1a CTR endogenously expressed in UMR106-06 cells with the cloned C1a CTR in transfected HEK 293 cells, in which expression is driven by a heterologous promoter. It was found that treatment of both cell lines with either salmon CT or human CT reduced the density of cell surface CTR in a dose- and time-dependent manner. However, the magnitude of the response was greater in UMR106-06 cells, and salmon CT was more potent than human CT in both cell lines. Recovery from down-regulation was rapid in transfected cells (< 2 h), but was comparatively delayed in UMR106-06 cells, where less than 70% of receptor-binding capacity had returned by 24 h. In both cell lines, treatment with either agonist increased the basal activity of CT-sensitive adenylate cyclase and caused a time-dependent reduction in the responsiveness of adenylate cyclase to a second challenge with CT. Reduced responsiveness occurred under conditions of minimal loss of CTR from the cell surface, consistent with an uncoupling of the receptor from the signal transduction apparatus. Recovery of CT-sensitive adenylate cyclase was complete in transfected cells by 24 h, but was delayed in UMR106-06 cells, paralleling the slow recovery of receptor binding. CT-induced down-regulation of the CTR was not mimicked by receptor-independent activation of protein kinase A or protein kinase C. However, treatment of cells for 24 h, but not for 4 h, with phorbol ester caused a partial loss of CTR binding in UMR106-06 cells and resulted in an approximately 200% increase in CTR binding in transfected HEK 293 cells. CTR messenger RNA levels, as assessed by reverse transcription-PCR, were not changed by any of the above treatments. These results suggest that CT-induced receptor down-regulation and modulation of the ability of CT to activate adenylate cyclase are inherent properties of the receptor, as they can be recapitulated in an otherwise CTR-naive cell line, in which receptor expression is driven by a heterologous gene promoter. Moreover, and in contrast with CTR regulation in osteoclasts, activation of protein kinase A is insufficient for ligand-induced regulation of the CTR in these nonosteoclastic cell lines, and receptor regulation does not appear to involve altered messenger RNA levels.
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PMID:Homologous regulation of the rat C1a calcitonin receptor (CTR) in nonosteoclastic cells is independent of CTR messenger ribonucleic acid changes and cyclic adenosine 3',5'-monophosphate-dependent protein kinase activation. 889 20

The D2 dopamine receptor is known to be functionally coupled to the inhibition of adenylate cyclase when expressed in a number of mammalian cell lines. However, functional coupling of the recently discovered D3 and D4 dopamine receptor subtypes has been more difficult to demonstrate. In this study, human D2, D3 and D4 receptors were stably expressed separately in human embryonic kidney cells (HEK 293). In these cells, activation of D2, D3 or D4 receptors resulted in the inhibition of forskolin-stimulated adenylate cyclase activity in a dose responsive manner. This activation was prevented by pre-incubation of the cells expressing these receptors with the dopaminergic antagonist haloperidol. Radioligand binding studies using [3H]spiperone confirmed that the atypical neuroleptic clozapine has higher affinity for the human D4 receptor than the D3 or D4 receptors, although only 6-fold higher than the D2 receptor in this study. In addition, ribonuclease protection studies demonstrated the presence of D4 dopamine receptor mRNA in human brain regions.
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PMID:Functional coupling of human D2, D3, and D4 dopamine receptors in HEK293 cells. 890 44

We have characterized the expression pattern and pharmacological profile of activation of metabotropic glutamate receptors (mGluRs) in immortalized, gonadotropin releasing hormone (GnRH)-secreting GT1-7 cells, which represent a homogeneous cellular population of hypothalamic origin. These cells are known to respond to the mGluR agonist (1S,3R)-cyclopentanedicarboxylic acid (1S,3R-ACPD) with increased GnRH release. To establish which specific mGluR subtypes are expressed by GT1-7 cells, we used polyclonal antibodies raised against non-conserved regions of the carboxy-terminal domains of individual subtypes. The selectivity of these antibodies was tested in HEK 293 cells transiently transfected with each mGluR subtype. GT1-7 cells stained positively for the subtypes mGluR1a, -1b and -5 (belonging to group I mGluR2/3 (group II) and mGluR7 (group III). Agonists of group I mGluRs, including 1S,3R-ACPD, activated phosphoinositide hydrolysis in GT1-7 cells. This effect, however, was manifested only when cell density was low, and it disappeared when cells reached confluence. Stimulation of phosphoinositide hydrolysis could not therefore have been related to hormone secretion because 1S,3R-ACPD effectively released GnRH in confluent cultures. We then focused on group II and III mGluRs, which in transfected cells are negatively linked to adenylate cyclase activity. Unexpectedly, however, agonist which preferentially activate group II and III mGluRs increased both basal and forskolin-stimulated cAMP accumulation in GT1-7 cells. Stimulation of cAMP accumulation by mGluR agonists was not prevented by enzymatic depletion of endogenous adenosine, but was obliterated when cells were incubated with agonists of receptors positively coupled to adenylate cyclase, such as beta-adrenergic and prostaglandin E2 receptors. These results suggest that GT1-7 cells express a novel mGluR subtype positively coupled to adenylate cyclase, which shares the same transduction pathway of other classical receptors coupled with a Gs-type of GTP-binding protein.
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PMID:Immortalized hypothalamic neurons express metabotropic glutamate receptors positively coupled to cyclic AMP formation. 895 Jan 4


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