Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5-7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nM) and [3H]XAC (4 nM), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5'. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were > 30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5'-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.
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PMID:Site-directed mutagenesis identifies residues involved in ligand recognition in the human A2a adenosine receptor. 777 60

Adenosine is thought to be a major effector in immunological stimulation of Cl- secretion in intestinal epithelia. Previous studies indicate that both apical and basolateral domains of intestinal epithelial cells possess functionally defined adenosine receptors. However, it is unclear whether the same receptor subclass is expressed, what the receptor subclass(es) is, or how the receptors signal the Cl- secretory response. We now characterize the intestinal epithelial adenosine receptor subtype using the model epithelium, T84. Both apical and basolateral adenosine receptor agonist response profiles revealed a hierarchy (ED50) of 5'-(N-ethylcarboxamido)adenosine > adenosine > CGS-21680. Similarly inhibition studies revealed identical ID50 hierarchies for apical and basolateral antagonism by xanthine amine congener > 1,3-diethyl-8-phenylxanthine > aminophylline. Analyses of both agonist and antagonist pharmacological hierarchies in Chinese hamster ovary cells stably expressing the A2b receptor revealed these same hierarchies. Northern blots performed on RNA extracted from polarized T84 monolayers demonstrated no detectable message for A1 or A2a adenosine receptor, but strong hybridization was detected for the A2b adenosine receptor. Subsequent Northern blots of RNA prepared from human alimentary tract revealed that A2b adenosine receptor message was heavily expressed throughout the colon, in the appendix, and more modestly expressed in the small intestine (ileum). Analyses of cAMP generation in T84 cells in response to adenosine indicated that the basolateral A2b receptor elicits Cl- secretion through this signaling pathway. Stimulation of Cl- secretion through the apical A2b receptor exhibited relatively small but significant increases in cAMP compared with basolateral stimulation. The protein kinase A inhibitor H-89, used at concentrations that did not affect short circuit current responses to the Ca(2+)-mediated agonist carbachol, effectively inhibited short circuit current elicited by either apical or basolateral adenosine. These data suggest that the major intestinal epithelial adenosine receptor is the A2b subclass, which is positively coupled to adenylate cyclase. Such observations have potentially important implications for the treatment of diarrheal diseases.
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PMID:The A2b adenosine receptor mediates cAMP responses to adenosine receptor agonists in human intestinal epithelia. 783 74

1. The effect of calcitonin gene-related peptide (CGRP) on [3H]-acetylcholine ([3H]-ACh) release from motor nerve endings and its interaction with presynaptic facilitatory A2a-adenosine and nicotinic acetylcholine receptors was studied on rat phrenic nerve-hemidiaphragm preparations loaded with [3H]-choline. 2. CGRP (100-400 nM) increased electrically evoked [3H]-ACh release from phrenic nerve endings in a concentration-dependent manner. 3. The magnitude of CGRP excitation increased with the increase of the stimulation pulse duration from 40 microseconds to 1 ms, keeping the frequency, the amplitude and the train length constants. With 1 ms pulses, the evoked [3H]-ACh release was more intense than with 40 microseconds pulse duration. 4. Both the nicotinic acetylcholine receptor agonist, 1,1-dimethyl-4-phenylpiperazinium, and the A2a adenosine receptor agonist, CGS 21680C, increased evoked [3H]-ACh release, but only CGS 21680C potentiated the facilitatory effect of CGRP. This potentiation was prevented by the A2a adenosine receptor antagonist, PD 115,199. 5. Adenosine deaminase prevented the excitatory effect of CGRP (400 nM) on [3H]-ACh release. This effect was reversed by the non-hydrolysable A2a-adenosine receptor agonist, CGS 21680C. 6. The nicotinic antagonist, tubocurarine, did not significantly change, whereas the A2-adenosine receptor antagonist, PD 115,199, blocked the CGRP facilitation. The A1-adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, potentiated the CGRP excitatory effect. 7. The results suggest that the facilitatory effect of CGRP on evoked [3H]-ACh release from rat phrenic motor nerve endings depends on the presence of endogenous adenosine which tonically activates A2a-adenosine receptors. Since both CGRP and A2a-adenosine receptors are positively coupled to the adenylate cyclase/cyclic AMP system, cooperation between these receptors might occur at the second messenger transduction system level.
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PMID:Potentiation by tonic A2a-adenosine receptor activation of CGRP-facilitated [3H]-ACh release from rat motor nerve endings. 800 2