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)

Several glucagon analogs containing substitutions for serine have been synthesized to assess the role of the four serine residues in the hormone. The strategic importance of His1 has been confirmed, and we have previously identified an aspartic acid critical for activity at position 9. While these findings have led to a series of pure glucagon antagonists, the details of specific glucagon-receptor interactions that switch on the ensuing signaling events are still not readily apparent. The requirement for serine was tested by the chemical synthesis of a series of analogs containing substitutions for the hydrophilic hydroxyl group in each of the highly conserved serine residues at positions 2, 8, 11, and 16 of glucagon. The resulting analogs were analyzed in rat hepatocyte membranes for their receptor-binding affinities as well as their abilities to stimulate adenylate cyclase. Positions 2 and 8 were the most sensitive to modification, where both binding and activity were adversely affected. This is consistent with the notion that although the sequence responsible for transduction lies in the amino-terminal half of glucagon, some residues at that end also contribute to binding affinity. Modifications at position 11 generated high-binding-affinity derivatives that were full or moderate agonists. In contrast, position 16 replacement analogs maintained significant receptor binding affinities while the agonist properties were almost completely lost, thus separating binding and transduction functions. Therefore, Ser16 is a third critical residue that determines glucagon activity. It is postulated, but not proven, that a serine residue, together with His1 and Asp9, may participate in the putative active center of glucagon, which, upon initial recognition and binding to receptor, leads to transduction of the biological signal. A dependence of the glucagon action on a three-residue cooperative mechanism might be analogous to the charge-relay scheme of serine proteases. It is suggested that, after binding to its receptor, glucagon becomes activated and functions like a coenzyme in catalyzing the specific hydrolysis of a peptide bond in the receptor, generating new amino and carboxyl end groups, and that one of these exposed chains may contact the GTP-binding protein and activate it for further interaction with adenylate cyclase. This idea was supported by inhibition experiments with 4-amidinophenylmethanesulfonyl fluoride (APMSF), a specific and irreversible inhibitor of serine proteases, which at a concentration of 5 mM completely suppressed cAMP formation by glucagon in liver membranes. cAMP formation was not affected if either glucagon or membranes were separately pretreated with APMSF and then assayed.
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PMID:Identification of an essential serine residue in glucagon: implication for an active site triad. 829 May 48

Point mutations in the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor have been shown to cause constitutive activation which results in precocious puberty in affected males. We introduced one of these mutations, Asp-556 --> Gly, into the rat LH/hCG receptor and demonstrated that the mutant receptor constitutively activated adenylate cyclase in transfected 293 T cells. The cell surface expression of the mutant receptor was lower than that of the wild type receptor. Pulse-chase studies showed that the 73-kDa precursor of both the mutant and wild type receptors was synthesized at comparable efficiencies. However, post-translational processing of the mutant receptor to the mature 92-kDa form, which has N-linked complex type oligosaccharide chains, was impaired. Sensitivity of the mutant receptor to peptide-N-glycanase F and endoglycosidase H, and insensitivity to sialidase indicated that the 73-kDa species represents the high mannose form that has not yet been trafficked through the medial and trans Golgi. Additionally, although the wild type receptor was palmitoylated, the mutant receptor was not. Although the high mannose 73-kDa species is capable of binding LH/hCG, our results show that post-translational processing in the Golgi is required for the mature 92-kDa receptor to reach the cell surface.
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PMID:Post-translational processing in the Golgi plays a critical role in the trafficking of the luteinizing hormone/human chorionic gonadotropin receptor to the cell surface. 903 11

Treatment of U937 cells with dolichyl phosphate led to an increase in the activity of the ICE family protease CPP32, accompanied with cleavage of pre-CPP32 to generate p17. Peptide inhibitors YVAD-cmk and Z-Asp-CH2-DCB (specific to ICE) and DEVD-CHO (specific to CPP32) blocked the dolichyl phosphate-induced apoptosis. The dolichyl phosphate-induced increase of CPP32 activity was inhibited by adenylate cyclase inhibitors, SQ 22536 and 2',5'-dideoxyadenosine. Dolichyl phosphate caused a transient increase of intracellular cAMP concentration. The results suggest that modulation of cAMP synthesis due to the stimulation of adenylate cyclase by dolichyl phosphate plays a critical role in CPP32 activation and apoptosis.
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PMID:CPP32 activation during dolichyl phosphate-induced apoptosis in U937 leukemia cells. 925 10

Extensive evidence gathered from structure-activity relationship analysis has identified and confirmed specific positions in the glucagon sequence that are important either for binding to its receptor or for signal transduction. Fifteen glucagon analogues have been designed and synthesized by incorporating structural changes in the N-terminal region of glucagon, in particular histidine-1, phenylalanine-6, and aspartic acid-9. This investigation was conducted to study the role of phenylalanine at position 6 on the glucagon mechanism of action. These glucagon analogues have been made by either deleting or substituting hydrophobic groups, hydrophilic groups, aromatic amino acids, or a D-phenylalanine residue at this position. The structures of the new analogues are as follows: [des-His1, des-Phe6, Glu9]glucagon-NH2 (1); [des-His1,Ala6,Glu9]glucagon-NH2 (2); [des-His1,Tyr6,Glu9]glucagon-NH2 (3); [des-His1,Trp6,Glu9]-glucagon-NH2 (4); [des-His1,D-Phe6,Glu9]glucagon-NH2 (5); [des-His1,Nle6,Glu9]glucagon-NH2 (6); [des-His1,Asp6,Glu9]glucagon-NH2 (7); [des-His1,des-Gly4,Glu9]glucagon-NH2 (8); [desPhe6,-Glu9]glucagon-NH2 (9); [des-Phe6]glucagon-NH2 (10); [des-His1, des-Phe6]glucagon-NH2 (11); [des-His1, des-Phe6,Glu9]glucagon (12); [des-Phe6,Glu9]glucagon (13); [des-Phe6]glucagon (14); and [des-His1, des-Phe6]glucagon (15). The receptor binding potencies IC50 values are 48 (1), 126 (2), 40 (3), 19 (4), 100 (5), 48 (6), 2000 (7), 52 (8), 113 (9), 512 (10), 128 (11), 1000 (12), 2000 (13), 500 (14), and 200 nM (15). All analogues were found to be antagonists unable to activate the adenylate cyclase system even at concentrations as high as 10(-5) M except for analogues 6 and 8, which were found to be weak partial agonists/partial antagonists with maximum stimulation between 6-12%. In competitive inhibition experiments, all the analogues caused a right shift of the glucagon-stimulated adenylate cyclase dose-response curve. The pA2 values were 8.20 (1), 6.40 (2), 6.20 (3), 6.25 (4), 6.30 (5), 6.30 (7), 6.05 (8), 6.20 (9), 6.30 (10), 6.25 (11), 6.10 (12), 6.20 (13), 6.20 (14), and 6.35 (15).
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PMID:The role of phenylalanine at position 6 in glucagon's mechanism of biological action: multiple replacement analogues of glucagon. 925 62

The human beta-adrenoceptor is a member of the seven-transmembrane family of receptors, encoded by a gene on chromosome 5. beta-Adrenoceptors have been classified into beta1, beta2, and beta3 subgroups, with beta2-receptors being widely distributed in the respiratory tract, particularly in airway smooth muscle. Intracellular signaling following beta2-adrenoceptor activation is largely affected through a trimeric Gs protein coupled to adenylate cyclase. Cyclic AMP (cAMP) induces airway relaxation through phosphorylation of muscle regulatory proteins and attenuation of cellular Ca2+ concentrations. Alternative cAMP-independent pathways involving activation of membrane maxi-K+ channels and coupling through Gi to the MAP kinase system have also been described. Site-directed mutagenesis has identified Asp 113 and Ser 204/207 within the third and fourth membrane domains as the active site of the beta2-receptor, critical for beta2-agonist binding and activity. beta2-Agonists have been characterized as those that directly activate the receptor (albuterol), those that are taken up into a membrane depot (formoterol), and those that interact with a receptor-specific auxiliary binding site (salmeterol). These differences in mechanism of action are reflected in the kinetics of airway smooth muscle relaxation and bronchodilation in patients with asthma. beta-Adrenoceptor desensitization associated with beta2-agonist activation is a consequence of phosphorylation by beta-ARK and uncoupling of the receptor from Gs following beta-arrestin binding, of internalization and recycling of the receptor through processes of sequestration and resensitization and downregulation, modulated by an effect on receptor gene expression. The degree of receptor desensitization appears to differ, depending on the cell or tissue type, and is reflected in the different profiles of clinical tolerance to chronic beta2-agonist therapy. A number of polymorphisms of the beta2-receptor have been described that appear to alter the behavior of the receptor following agonist exposure. These include Arg-Gly 16, Glu-Gln 27, and Thr-lle 164. The Gly 16 receptor downregulates to a greater extent and is associated with increased airway hyperreactivity, nocturnal symptoms, and more severe asthma. The Glu 27 form appears to protect against downregulation and is associated with less reactive airways. An individual can be homozygous or heterozygous for given polymorphisms, and large populations will have to be studied to determine their importance to the asthma phenotype.
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PMID:The beta-adrenoceptor. 981 38

This review examines the recent progress in the field of angiotensin receptors. Multiplicity of these receptors was demonstrated initially on the basis of pharmacologic differences and then confirmed by expression cloning. AT1 receptors are predominant in the adult. They are widely distributed and mediate all of the known biologic effects of angiotensin II (AngII) through a variety of signal transduction systems, including activation of phospholipases C and A2, inhibition of adenylate cyclase, opening of calcium channels, and activation of tyrosine kinases. AT2 receptors are predominant in the fetus, but also present in adult tissues such as the adrenals, ovaries, uterus, and brain. AngII via these receptors exerts effects often opposed to those mediated by the AT1 receptors. Signal transduction implicates protein tyrosine phosphatase stimulation. AT1 and AT2 receptor expressions are regulated differently, and regulation is also tissue-specific. AT1 and AT2 receptors have been demonstrated in endothelial cells. Activation of AT1 receptors results in production of vasodilatory agents, nitric oxide, and prostacyclin (PGI2), which counteract the direct vasoconstrictor effects of Ang II on the adjacent smooth muscle cells. AT1 receptors on mesangial cells, smooth muscle cells, and fibroblasts are involved in cell growth and fibrosis, the latter being due both to an increase in the synthesis and a decrease in the degradation of the main components of the extracellular matrix. These AT1 receptor-dependent effects are for the most part indirect and mediated by growth factors, cytokines, and other peptides, including endothelin, transforming growth factor-beta1, and platelet-derived growth factor. AngII is metabolized into active fragments by deletion of the terminal amino acids on both ends. AngIII and AngIV are formed by successive deletions of aspartic acid and arginine at the N terminus. AngII (1-7) is obtained by deletion of phenylalanine at the C terminus. AngIII shares the same receptors and exerts the same effects as AngII. AngIV and AngII (1-7) recognize the AT1 and AT2 receptors with a lesser affinity than AngII and, in addition, possess their own receptors that mediate effects often opposed to those of AngII.
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PMID:Angiotensin II receptors. 989 38

To assess the role of the conserved DPWXY motif of the seventh transmembrane domain in prostanoid receptor-mediated G protein activation, we have mutated the negatively charged Asp-318 in this motif of the Gi-coupled mouse prostaglandin EP3 receptor to uncharged but polar Asn (EP3-D318N) and to the non-polar Leu (EP3-D318L). The EP3 agonist and antagonist showed similar binding affinities for the wild-type and two mutant receptors. The wild-type and EP3-D318N receptors but not EP3-D318L receptor associated with Gi in guanine nucleotide- and pertussis toxin-sensitive manners. On the other hand, the wild-type receptor but not two mutant receptors had the ability to stimulate GTPase activity and to inhibit the adenylate cyclase. These findings demonstrate that the chemical nature of the amino acid residue at position 318 of the seventh transmembrane domain of the EP3 receptor dissociates the step of Gi association from that of subsequent Gi activation in the process of the EP3 receptor-Gi coupling.
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PMID:The key amino acid residue of prostaglandin EP3 receptor for governing G protein association and activation steps. 1008 73

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

Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with G(s)alpha. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor and fusion proteins generated between this form of the receptor and the alpha subunits of its cognate G-protein G(s), G(i1), a G-protein which it fails to activate in co-expression studies, and a chimaeric G(i1)-G(s)6 (a form of G(i1) in which the C-terminal six amino acids were replaced with the equivalent sequence of G(s)) were stably expressed in HEK293 cells. These were detected by [(3)H]ligand-binding studies and by immunoblotting with both an anti-FLAG antibody and with appropriate antisera to the G-proteins. Each construct displayed similar affinity to bind the agonist iloprost. Iloprost stimulated adenylate cyclase activity in clones expressing both IP prostanoid receptor and the IP prostanoid receptor-G(s)alpha fusion protein, and both constructs were shown to interact with and activate endogenously expressed G(s)alpha. Addition of iloprost to membranes of cells expressing the isolated receptor resulted in a small stimulation of high-affinity GTPase activity. Iloprost produced no stimulation of GTPase activity which could be attributed to the IP prostanoid receptor-G(i1)alpha fusion. However, the fusion proteins containing either G(s)alpha or G(i1)-G(s)6alpha produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-G(i1)-G(s)6alpha fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normalization of such results for levels of expression of the IP prostanoid receptor constructs demonstrated a 5-fold higher stimulation of GTPase activity when using the G(s)alpha-containing fusion protein and a 9-fold improvement when using the fusion protein containing G(i1)-G(s)6alpha to detect G-protein activation compared with expression of the isolated receptor.
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PMID:Analysis of agonist function at fusion proteins between the IP prostanoid receptor and cognate, unnatural and chimaeric G-proteins. 1045 34

Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.
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PMID:Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280). 1069 83


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