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)

Gsalpha regulates the differentiation of 3T3-L1 mouse embryonic fibroblasts to adipocytes, a process termed adipogenesis. Through the expression of chimera created by substituting regions of Gsalpha with corresponding regions of the G protein Gialpha2, the domain of Gsalpha involved in repression of adipogenesis was localized to sequence 146-235 of the molecule (Wang, H-y., Johnson, G. L., Liu, X. , Malbon, C. C. (1996) J. Biol. Chem. 271, 22022-22029). As a prelude to alanine-scanning mutagenesis, chimeras in Gsalpha constructed from trisection of the sequence 125-213 of Gialpha2 were expressed stably, and clones were evaluated for the ability of the chimera to repress adipogenesis in response to the inducers, dexamethasone and methylisobutylxanthine, in combination. The chimera containing sequence 150-177 of Gialpha2 repressed adipogenesis, whereas the chimeras with either sequence 125-149 or 178-213 of Gialpha2 failed to repress induction of adipogenesis. Alanine-scanning mutagenesis of these two critical domains was performed first in clusters and then confirmed by analysis of single mutations. Six residues unique to Gsalpha were identified as critical to repression of adipogenesis, Asn167, Cys200, Leu203, Ser205, Val214, and Lys216. Leu203 and Ser205 are required in tandem, as mutagenesis to alanine of either one alone was without effect on repressor activity. The remaining four residues are required for repressor activity; mutation of any one of these abolishes the ability of Gsalpha to repress adipogenesis, although not affecting the ability of the mutant form of Gsalpha to regulate adenylylcyclase. Using conserved landmarks found in the crystal structures of Gialpha1 and Gsalpha, the Leu203 and Ser205 cluster appears to be exposed, closely aligned and located in switch I region. Asn167, Val214, and Lys216 project to regions on Gsalpha that are exposed in the GTPgammaS-liganded state of the alpha subunit. We speculate that these residues constitute an important contact domain between Gsalpha and the effector controlling adipogenesis, which is yet to be identified.
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PMID:Identification of amino acid residues of Gsalpha critical to repression of adipogenesis. 956 89

Dopamine D2 receptors contain a cluster of serine residues in the fifth transmembrane domain that contribute to activation of the receptor as well as to the binding of agonists. We used rat D2S dopamine receptor mutants, each containing a serine-to-alanine substitution (S193A, S194A, S197A), to investigate the mechanism through which these residues affect activation of the receptor. Activation of the mutant receptor S194A was abolished in an agonist-dependent manner, such that dopamine no longer inhibited cAMP accumulation in C6 glioma cells or activated G protein-regulated K+ channels in Xenopus laevis oocytes, whereas the efficacy of several other agonists was unaffected. Dihydrexidine did not inhibit cAMP accumulation at either S193A or S194A. The decreased efficacy of dihydrexidine at S193A and S194A and dopamine at S194A was associated with a decreased ability to detect a GTP-sensitive high affinity binding state for these agonists. The ability of dopamine to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate binding via S194A also was decreased by approximately 50%. Finally, constitutive stimulation of [35S]guanosine-5'-O-(3-thio)triphosphate binding and inhibition of adenylate cyclase by the D2S receptor was reduced by mutation of either S193 or S194. These data support the existence of multiple active receptor conformations that are differentially sensitive to mutation of serine residues in the fifth-transmembrane domain.
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PMID:Contribution of serine residues to constitutive and agonist-induced signaling via the D2S dopamine receptor: evidence for multiple, agonist-specific active conformations. 968 86

A sequence motif of 20 amino acid residues within the C-terminal portion of the rat somatostatin receptor subtype 4 (SSTR4) has been shown to prevent rapid agonist-dependent receptor internalization in transfected human embryonic kidney (HEK) cells. Molecular dissection of this motif by biochemical ligand-binding assays revealed that the block was released by mutating a single residue (threonine 331) to an alanine. These data are in line with confocal microscopic analysis of cultured primary neurons microinjected with cDNA constructs encoding either SSTR4 or the mutant T331A. Immunocytochemical analysis showed that the mutant receptor, but not SSTR4, was internalized. However, internalized T331A was not recycled to the cell surface, suggesting that it lacks sequence elements that determine intracellular sorting after endocytosis. Neither wildtype SSTR nor the mutant T331A exhibited functional desensitization when assayed for their ability to inhibit adenylate cyclase. In agreement with this, the wt receptor and its mutant were not phosphorylated in response to agonist treatment. Lack of desensitization of SSTR4 has been electrophysiologically verified by coexpressing the receptor with a G-protein-gated, inwardly rectifying potassium channel in Xenopus oocytes. A strong somatostatin 14 (SST14)-activated inward potassium current was observed that was long-lasting and which decayed only slowly after washout of the agonist. This is in contrast to another somatostatin receptor subtype, SSTR3, which mediates rapidly desensitizing currents. Binding experiments on HEK cells transfected with either SSTR3 or 4 indicated that this difference is not attributable to slow dissociation of the agonist from the receptor, suggesting that SSTR4 mediates long-lasting signalling, a property which may be relevant for clinical therapy.
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PMID:Rat somatostatin receptor subtype 4 can be made sensitive to agonist-induced internalization by mutation of a single threonine (residue 331). 980 48

Somatostatin agonists are rapidly and efficiently internalized with the somatostatin sst2 receptor. The fate of internalized agonists and receptors is of critical importance because the rate of ligand recycling back to the cell surface can limit the amount of radioligand accumulated inside the cells, whereas receptor recycling might be of vital importance in providing the cell surface with dephosphorylated, resensitized receptors. Furthermore the accumulation of radioisotope-conjugated somatostatin agonists inside cancer cells resulting from receptor-mediated internalization has been used as a treatment for cancers that overexpress somatostatin receptors. In the present study, radio-iodinated agonists at the sst2 somatostatin receptor were employed to allow quantitative analysis of the fate of endocytosed agonist. After endocytosis, recycling back to the cell surface was the main pathway for both 125I-labelled somatostatin-14 (SRIF-14) and the more stable agonist 125I-labelled cyclo(N-Me-Ala-Tyr-d-Trp-Lys-Abu-Phe) (BIM-23027; Abu stands for aminobutyric acid), accounting for 75-85% of internalized ligand when re-endocytosis of radioligand was prevented. We have shown that there is a dynamic cycling of both somatostatin agonist ligands and receptors between the cell surface and internal compartments both during agonist treatment and after surface-bound agonist has been removed, unless steps are taken to prevent the re-activation of receptors by recycled agonist. Internalization leads to increased degradation of 125I-labelled SRIF-14 but not 125I-labelled BIM-23027. The concentration of recycled agonist accumulating in the extracellular medium was sufficient to re-activate the receptor, as measured both by the inhibition of forskolin-stimulated adenylate cyclase and the recovery of surface receptor number after internalization.
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PMID:Fates of endocytosed somatostatin sst2 receptors and associated agonists. 982 Aug 3

The human vasoactive intestinal peptide/pituitary adenylate cyclase activating peptide receptor1 (VPAC1) belongs to the class II subfamily of G protein-coupled receptors. Specific changes by mutagenesis of a strictly conserved threonine (H) into lysine (K), proline (P) or alanine (A) at position 343 of the human VPAC1 receptor resulted in its constitutive activation with respect to cAMP production. Transfection of these mutants into Cos cells evoked a 3.5 fold-increase in the cAMP level as compared to cells transfected with the wild-type receptor. In contrast other mutants such as T343C, T343E or T343F were not constitutively activated. They were otherwise expressed at the cell surface of transfected nonpermeabilized cells. Double mutants were then constructed in which the T343K mutation was associated with a point mutation in the the N-terminal extracellular domain that totally abolished VIP binding or VIP-stimulated cAMP production i.e. E36A or D68A. The corresponding double mutants T343K-E36A and T343K-D68A were no longer constitutively activated. A control double mutant (T343K-D132A) with an unaltered dissociation constant for VIP and cAMP response to VIP, was still constitutively activated. Our findings demonstrate that constitutive activation of the VPAC1 receptor can be evoked by specific mutations of T343 at the junction of the second intracellular loop and fourth transmembrane segment. This constitutive activation appears to require the functional integrity of the N-terminal extracellular VIP binding domain.
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PMID:The human vasoactive intestinal Peptide/Pituitary adenylate cyclase activating peptide receptor 1 (VPAC1): constitutive activation by mutations at threonine 343. 992 Jul 25

Despite many triumphs, a significant limitation of the usefulness of many of the available B-cell lines for the study of insulin secretion are either inappropriate or lack of responsiveness to glucose. Commonly employed cell lines generated prior to the 1990s following X-ray irradiation (RINm5F cells) or simian virus 40 B-cell transformation (HIT-T15 cells and BTC) fall into this category. More recent success has been achieved with the generation of INS-1 cells and MIN6 cells, but the production of these cell lines owes much to good fortune, dedication and hard work. In the present era, molecular biology techniques provide the opportunity to engineer novel pancreatic B-cell lines which possess many attributes of normal insulin-secreting cells. This review describes the electrofusion of normal NEDH rat pancreatic B-cells with immortal RINm5F cells to create three new glucose-responsive clonal insulin-secreting cells, designated BRIN-BG5, BRIN-BG7 and BRIN-BD11. These cell lines exhibit up to four-fold insulin-secretory responses to depolarization with 25 mmol/l K+, 7.68 mmol/l Ca2+, 10 mmol/l L-alanine, and activation of protein kinase C or adenylate cyclase with 10 nmol/l phorbol- 12-myristate-13-acetate or 25 micromol/l forskolin, respectively. The maximal insulin-secretory response of both BRIN-BG5 and BRIN-BG7 cells to glucose occurred at 8.4 mmol/l (1.9- and 1.8-fold increases, respectively). In contrast, 4.2-16.7 mmol/l glucose evoked a stepwise 2- to 3-fold of insulin release from BRIN-BD11 cells. The superior glucose responsiveness of BRIN-BD11 cells compared with BRIN-BG5 or BRIN-BG7 cells was associated with increased expression of GLUT-2 and a greater contribution of glucokinase to total glucose phosphorylating enzyme activity. Furthermore, BRIN-BD11 cells also showed appropriate responses to a diverse range of modulators of pancreatic B-cell function, including amino acids, neurotransmitters and sulphonylurea drugs. Collectively these observations indicate that genetic modification of insulin-secreting cells by electrofusion (or transfection with cDNA) offers a new avenue for generation of useful clonal glucose-responsive pancreatic B-cell lines for studies of insulin secretion and transplantation in insulin-dependent diabetes mellitus.
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PMID:Engineering cultured insulin-secreting pancreatic B-cell lines. 993 Sep 71

The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains. In the present work, CyaC was produced in Escherichia coli as a histidine-tagged recombinant protein and purified to homogeneity. CyaC showed ability to autophosphorylate in vitro with the gamma-phosphate of [gamma-32P]ATP. CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His572) and receiver domains (Asp60 or Asp895) were replaced by glutamine and alanine residues, respectively. After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined. It was found that His572 is the initial phosphorylation site and that the phosphoryl group once bound to His572 is transferred to Asp895. The enzyme activities of the CyaC derivatives defective in His572 or Asp895 were considerably reduced. Asp895 is phosphorylated by acetyl [32P]phosphate, a small phosphoryl molecule, but Asp60 is not. Acetyl phosphate stimulates adenylate cyclase activity only when Asp895 is intact. These results suggest that the phosphorylation of Asp895 is essential for the activation of adenylate cyclase and that Asp60 functions differently from Asp895 in regulating the enzyme activity.
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PMID:Activation of a cyanobacterial adenylate cyclase, CyaC, by autophosphorylation and a subsequent phosphotransfer reaction. 1032 24

The secretin amino terminal residues are essential for high affinity binding to the cognate receptor and for the subsequent activation of adenylate cyclase. It has been already established that two basic residues of the receptor TM 2 are involved in the interaction with aspartate 3 of the ligand. The present work investigated the hypothesis that two conserved tyrosine residues of the TM 1 (Tyrosines 124 and 128) could also participate to the positioning of the amino terminus of the ligand. Tyrosines 124 and 128 were mutated into alanine and histidine residues, and the properties of the mutant receptors, expressed in CHO cells, were compared with those of the wild-type receptor. Mutation of tyrosine 124 to Ala or His decreased the affinity of the receptor for secretin, [Glu3]secretin, [Asn3]secretin and the secretin fragment 2-27, and reduced the intrinsic activity of [Asn3]secretin. Mutation of tyrosine 128 to Ala, but not to His reduced 50-fold secretin and [Asn3]secretin affinity but only 3-fold that of [Glu3]secretin. Secretin and [Glu3]Sn were equipotent in that mutant receptor. These results suggested that tyrosine 128 of the secretin receptor interacted directly with the [Asp3] residue of secretin and thus that the amino terminal domain of secretin interacts with amino acids buried in both the TM 1 and TM 2 helices.
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PMID:Mutations of aromatic residues in the first transmembrane helix impair signalling by the secretin receptor. 1041 23

Differences in the specificity of coupling of delta-opioid receptor with G-protein have been reported in the literature. We have observed a differential desensitization of delta-opioid receptors, endogenously expressed in the neuroblastoma cell line SK-N-BE, induced by peptide and alkaloid agonists. By combining photoaffinity labelling of receptor-activated G-proteins with [alpha-(32)P]azidoanilide-GTP and an anti-sense oligodeoxynucleotide strategy, we examined whether the chemical nature of opioid agonists, alkaloid or peptide, has a critical role in determining a G(i)alpha/G(o)alpha-protein-selective activation by the human delta-opioid receptors. Etorphine, a non-selective alkaloid agonist, was shown to stimulate the incorporation of [alpha-(32)P]azidoanilide-GTP into G(i)alpha1, G(i)alpha2, G(i)alpha3 and pertussis-toxin-insensitive Galpha subunits. In contrast, [d-Pen(2),d-Pen(5)]enkephalin (DPDPE; Pen is penicillamine) and Tyr-d-Ala-Phe-Asp-Val-Val-Gly-NH(2) (deltorphin I), selective peptide agonists, mainly activated G(i)alpha2 and G(o)alpha2 subunits. The 'knock-down' of G(o)alpha2 subunits by anti-sense oligodeoxynucleotides selectively decreased the inhibition of adenylate cyclase induced by DPDPE and deltorphin I, whereas anti-sense oligodeoxynucleotides directed against G(i)alpha2 subunits only decreased the potency of etorphine in inhibiting cAMP accumulation. These results suggest that the nature of the agonist, peptide or alkaloid is critical in determining the interaction between human delta-opioid receptors and Galpha subunits.
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PMID:Differential G-protein activation by alkaloid and peptide opioid agonists in the human neuroblastoma cell line SK-N-BE. 1043 2

We have characterised the organisation of genes encoding the glutamate and alanine rich protein (GARP) surface coat of the procyclic and epimastigote stages of Trypanosoma congolense in the tsetse fly. The GARP genes are arranged at two, possibly physically linked, loci, one of which exhibits allelic variation. One locus contains a single GARP gene, whilst both alleles of the other have a large tandem array of polycistronically transcribed GARP genes. Sequence analysis has revealed that there are very few coding differences between different GARP genes. A sequence related to the Trypanosoma brucei expression site associated gene 4 (encoding a transmembrane protein with a cytoplasmic adenylate cyclase domain) has been identified within a region at the downstream flank of one locus. There is no evidence that, within the single trypanosome, GARP genes are as diverse as the procyclin genes that encode a corresponding coat in T. brucei.
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PMID:Characterisation of the loci encoding the glutamic acid and alanine rich protein of Trypanosoma congolense. 1069 50


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