The Novel HLA-DRB1*12:113 Allele Was Identified by Next-Generation Sequencing.
HLA-DRB1*12:113 differs from HLA-DRB1*12:01:01:14 at codon 95 (GTC>ATC) and codon 191 (CGG>TGG).
Humans↗
Biomedical subjects
Soo-Kyung Kim
Publications and source records attributed to Soo-Kyung Kim.
The Novel HLA-DPB1*1650:01 N Allele Was Identified by Next-Generation Sequencing.
HLA-DPB1*1650:01 N differs from HLA-DPB1*135:01:01:01 in exon 2 codon 62 (CAG>TAG), introducing a premature stop codon.
Humans↗
The Novel HLA-C*07:1193 Allele Was Identified by Next-Generation Sequencing.
HLA-C*07:1193 differs from HLA-C*07:02:01:03 in exon 5 codon 292 (GCT>GTT).
Humans↗
Multiple residues in the second extracellular loop are critical for M3 muscarinic acetylcholine receptor activation.
Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys(220) residue, Gln(207), Gly(211), Arg(213), Gly(218), Ile(222), Phe(224), Leu(225), and Pro(228) were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance.
Amino Acid Sequence↗
Rapid identification of functionally critical amino acids in a G protein-coupled receptor.
G protein-coupled receptors (GPCRs) comprise one of the largest protein families found in nature. Here we describe a new experimental strategy that allows rapid identification of functionally critical amino acids in the rat M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR. This approach involves low-frequency random mutagenesis of the entire M3R coding sequence, followed by the application of a new yeast genetic screen that allows the recovery of inactivating M3R single point mutations. The vast majority of recovered mutant M3Rs also showed substantial functional impairments in transfected mammalian (COS-7) cells. A subset of mutant receptors, however, behaved differently in yeast and mammalian cells, probably because of the specific features of the yeast expression system used. The screening strategy described here should be applicable to all GPCRs that can be expressed functionally in yeast.
Amino Acid Sequence↗
2-triazole-substituted adenosines: a new class of selective A3 adenosine receptor agonists, partial agonists, and antagonists.
"Click chemistry" was explored to synthesize two series of 2-(1,2,3-triazolyl)adenosine derivatives (1-14). Binding affinity at the human A(1), A(2A), and A(3)ARs (adenosine receptors) and relative efficacy at the A(3)AR were determined. Some triazol-1-yl analogues showed A(3)AR affinity in the low nanomolar range, a high ratio of A(3)/A(2A) selectivity, and a moderate-to-high A(3)/A(1) ratio. The 1,2,3-triazol-4-yl regiomers typically showed decreased A(3)AR affinity. Sterically demanding groups at the adenine C2 position tended to reduce relative A(3)AR efficacy. Thus, several 5'-OH derivatives appeared to be selective A(3)AR antagonists, i.e., 10, with 260-fold binding selectivity in comparison to the A(1)AR and displaying a characteristic docking mode in an A(3)AR model. The corresponding 5'-ethyluronamide analogues generally showed increased A(3)AR affinity and behaved as full agonists, i.e., 17, with 910-fold A(3)/A(1) selectivity. Thus, N(6)-substituted 2-(1,2,3-triazolyl)adenosine analogues constitute a novel class of highly potent and selective nucleoside-based A(3)AR antagonists, partial agonists, and agonists.
Adenosine↗
Docking studies of agonists and antagonists suggest an activation pathway of the A3 adenosine receptor.
Structural determinants of ligand efficacy in the human A(3) adenosine receptor (AR) were studied using pharmacophore and docking analyses of various categories of A(3) selective ligands: inverse agonist, neutral antagonist (nonnucleoside and nucleoside), and agonist (partial and full). The homology modeling of GPCRs was adapted to provide two templates: the rhodopsin-based resting state for antagonist binding and a putative Meta I state, conformationally altered at a key residue (W6.48), for agonist binding. The preferential binding domains and/or local conformational changes associated with docking of three high affinity A(3)AR ligands were compared: inverse agonist PSB-11 1 ((R)-8-ethyl-4-methyl-2-phenyl-imidazo[2,1-i]purin-5-one); neutral antagonist MRE-3008F20 7 (5-[[(4-methoxyphenyl)amino]carbonyl]amino-8-methyl-2-(2-furyl)pyra-zolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine), and full agonist Cl-IB-MECA 21 (2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine) to define a distinct recognition mode for each. Ribose-containing agonists were more hydrophilic than nonnucleoside antagonists, and H-bonding ability at the ribose 3'- and 5'-positions was required for agonism. From the receptor perspective, common requirements for activation included the destabilization of H-bond networks at W6.48 and H7.43, the specific interactions of the ribose moiety in its putative hydrophilic pocket at T3.36, S7.42, and H7.43, the stabilization of the complex by inward movement of F5.43, and the characteristic rotation of W6.48. By analogy, outward rotation of the W6.48 side-chain upon activation of an internally-crosslinking mutant M(3) muscarinic receptor was indicated by constrained molecular dynamics (MD). Our results are consistent with an anti-clockwise rotation (from the extracellular view) of transmembrane domains 3, 5, 6, and 7, as proposed for other Family A GPCRs. Thus, the putative conformational changes associated with A(3)AR activation indicate a shared mechanism of GPCR activation similar to rhodopsin.
Adenosine↗
Orthogonal activation of the reengineered A3 adenosine receptor (neoceptor) using tailored nucleoside agonists.
An alternative approach to overcome the inherent lack of specificity of conventional agonist therapy can be the reengineering of the GPCRs and their agonists. A reengineered receptor (neoceptor) could be selectively activated by a modified agonist, but not by the endogenous agonist. Assisted by rhodopsin-based molecular modeling, we pinpointed mutations of the A(3) adenosine receptor (AR) for selective affinity enhancement following complementary modifications of adenosine. Ribose modifications examined included, at 3': amino, aminomethyl, azido, guanidino, ureido; and at 5': uronamido, azidodeoxy. N(6)-Variations included 3-iodobenzyl, 5-chloro-2-methyloxybenzyl, and methyl. An N(6)-3-iodobenzyl-3'-ureido adenosine derivative 10 activated phospholipase C in COS-7 cells (EC(50) = 0.18 microM) or phospholipase D in chick primary cardiomyocytes, both mediated by a mutant (H272E), but not the wild-type, A(3)AR. The affinity enhancements for 10 and the corresponding 3'-acetamidomethyl analogue 6 were >100-fold and >20-fold, respectively. 10 concentration-dependently protected cardiomyocytes transfected with the neoceptor against hypoxia. Unlike 10, adenosine activated the wild-type A(3)AR (EC(50) of 1.0 microM), but had no effect on the H272E mutant A(3)AR (100 microM). Compound 10 was inactive at human A(1), A(2A), and A(2B)ARs. The orthogonal pair comprising an engineered receptor and a modified agonist should be useful for elucidating signaling pathways and could be therapeutically applied to diseases following organ-targeted delivery of the neoceptor gene.
Adenosine A3 Receptor Agonists↗
Computational prediction of homodimerization of the A3 adenosine receptor.
Increasing evidence suggests that G protein-coupled receptors form functional dimers or larger oligomeric complexes through homo- or heterodimerization, and that various transmembrane (TM) domains contribute dimerization interfaces. In this study, monomeric receptor structures - either the monomeric crystallographic structure of bovine rhodopsin or an A(3) adenosine receptor (AR) homology model - were dimerized by computational methods assuming various TM contact regions, optimized, and compared. The semi-empirical oligomeric structure of mouse rhodopsin studied in a native disc membrane with atomic force microscopy was used to establish the distance between monomers in the initial dimeric models. Among eight variations of symmetrical homodimers of bovine rhodopsin, the favored dimeric assembly closely resembled the semi-empirical model, in which TM domains 4 and 5 were the contact site, thus validating this approach. We used similar methods to generate eight homodimers of the A(3)AR and found the favored dimeric interface similarly to be TM4-5. By this method, dimeric variations - TM1-2, TM2-3, TM2-4, TM3-4, TM4-5, TM5-6, TM6-7, and TM7-1 - were constructed with the SYBYL 7.0 program by using a novel "fit-centroids-normal" method. Fitting atoms considered one of eight TM-TM centroids or seven-TM centroids, two centroids of each monomer, and a normal atom passing through the plane containing all centroids. Following molecular dynamics, the most energetically favorable contact modes were identified. In addition to TM4-5, which was favored in both rhodopsin and A(3)AR dimeric models, TM1-2 dimers in which helices 8 also contacted each other were judged favorable. The largest contact surface area between the monomers among the various homodimers, determined by van der Waals calculation with the MOLCAD surface program, was for the TM4-5 dimer. This contact surface also showed a high degree of shape complementarity. In addition, the TM4-5 dimers made by this theoretical method were more stable than the semi-empirically determined dimer.
Animals↗
Action of nucleosides and nucleotides at 7 transmembrane-spanning receptors.
Ribose ring-constrained nucleosides and nucleotides to act at cell-surface purine recesptors have been designed and synthesized. At the P2Y1 nucleotide receptor and the A3 adenosine receptor (AR) the North envelope conformation of ribose is highly preferred. We have applied mutagenesis and rhodopsin-based homology modeling to the study of purine receptors and used the structural insights gained to assist in the design of novel ligands. Two subgroups of P2Y receptors have been defined, containing different sets of cationic residues for coordinating the phosphate groups. Modeling/mutagenesis of adenosine receptors has focused on determinants of intrinsic efficacy in adenosine derivatives and on a conserved Trp residue (6.48) which is involved in the activation process. The clinical use of adenosine agonists as cytoprotective agents has been limited by the widespread occurrence of ARs, thus, leading to undesirable side effects of exogenously administered adenosine derivatives. In order to overcome the inherent nonselectivity of activating the native receptors, we have introduced the concept of neoceptors. By this strategy, intended for eventual use in gene therapy, the putative ligand binding site of a G protein-coupled receptor is reengineered for activation by synthetic agonists (neoligands) built to have a structural complementarity. Using a rational design process we have identified neoceptor-neoligand pairs which are pharmacologically orthogonal with respect to the native species.
Animals↗
Conversion of A3 adenosine receptor agonists into selective antagonists by modification of the 5'-ribofuran-uronamide moiety.
The highly selective agonists of the A(3) adenosine receptor (AR), Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine), and its 4'-thio analogue, were successfully converted into selective antagonists simply by appending a second N-methyl group on the 5'-uronamide position. The 2-chloro-5'-(N,N-dimethyl)uronamido analogues bound to, but did not activate, the human A(3)AR, with K(i) values of 29 nM (4'-O) and 15 nM (4'-S), showing >100-fold selectivity over A(1), A(2A), and A(2B)ARs. Competitive antagonism was demonstrated by Schild analysis. The 2-(dimethylamino)-5'-(N,N-dimethyl)uronamido substitution also retained A(3)AR selectivity but lowered affinity.
Adenosine↗
Recovery of H2SO4 from waste acid solution by a diffusion dialysis method.
A diffusion dialysis method using anion exchange membrane was used to recover H2SO4 from waste sulfuric acid solution produced at the diamond manufacturing process. Effects of flow rate, operation temperature, and metal ion concentration on the recovery of H2SO4 were investigated. The recovery of H2SO4 increased with the concentration of H2SO4 and operation temperature. It also increased with the flow rate ratio of water/H2SO4 solution up to 1, above which no further increase was observed. The flow rate did not affect the rejection of Fe and Ni ions. About 80% of H2SO4 could be recovered from waste sulfuric acid which contained 4.5M free-H2SO4 at the flow rate of 0.26x10(-3) m3/hm3. The concentration of recovered H2SO4 was 4.3M and the total impurity was 2000 ppm. Preliminary economic evaluation has revealed that the dialysis system is highly attractive one that has payback period of only few months.
Antiporters↗
Identification of an agonist-induced conformational change occurring adjacent to the ligand-binding pocket of the M(3) muscarinic acetylcholine receptor.
To study the conformational changes that convert G protein-coupled receptors (GPCRs) from their resting to their active state, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system. Specifically, we employed a recently developed in situ disulfide cross-linking strategy that allows the formation of disulfide bonds in Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. At present, little is known about the conformational changes that GPCR ligands induce in the immediate vicinity of the ligand-binding pocket. To address this issue, we generated 11 Cys-substituted mutant M(3) muscarinic receptors and characterized these receptors in transfected COS-7 cells. All analyzed mutant receptors contained an endogenous Cys residue (Cys-532(7.42)) located within the exofacial segment of transmembrane domain (TM) VII, close to the agonist-binding site. In addition, all mutant receptors harbored a second Cys residue that was introduced into the exofacial segment of TM III, within the sequence Leu-142(3.27)-Asn-152(3.37). Disulfide cross-linking studies showed that muscarinic agonists, but not antagonists, promoted the formation of a disulfide bond between S151(3.36)C and Cys-532. A three-dimensional model of the inactive state of the M(3) muscarinic receptor indicated that Cys-532 and Ser-151 face each other in the center of the TM receptor core. Our cross-linking data therefore support the concept that agonist activation pulls the exofacial segments of TMs VII and III closer to each other. This structural change may represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface. To the best of our knowledge, this is the first direct demonstration of a conformational change occurring in the immediate vicinity of the binding site of a GPCR activated by a diffusible ligand.
Amino Acid Sequence↗
Pronounced conformational changes following agonist activation of the M(3) muscarinic acetylcholine receptor.
The conformational changes that convert G protein-coupled receptors (GPCRs) activated by diffusible ligands from their resting into their active states are not well understood at present. To address this issue, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system, employing a recently developed disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. In the present study, we generated and analyzed 30 double Cys mutant M(3) receptors, all of which contained one Cys substitution within the C-terminal portion of transmembrane domain (TM) VII (Val-541 to Ser-546) and another one within the C-terminal segment of TM I (Val-88 to Phe-92). Following their transient expression in COS-7 cells, all mutant receptors were initially characterized in radioligand binding and second messenger assays (carbachol-induced stimulation of phosphatidylinositol hydrolysis). This analysis showed that all 30 double Cys mutant M(3) receptors were able to bind muscarinic ligands with high affinity and retained the ability to stimulate G proteins with high efficacy. In situ disulfide cross-linking experiments revealed that the muscarinic agonist, carbachol, promoted the formation of cross-links between specific Cys pairs. The observed pattern of disulfide cross-links, together with receptor modeling studies, strongly suggested that M(3) receptor activation induces a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the cytoplasmic ends of TM I and VII. These findings should be of relevance for other family A GPCRs.
Amino Acid Sequence↗
(N)-methanocarba 2,N6-disubstituted adenine nucleosides as highly potent and selective A3 adenosine receptor agonists.
A series of ring-constrained (N)-methanocarba-5'-uronamide 2,N(6)-disubstituted adenine nucleosides have been synthesized via Mitsunobu condensation of the nucleobase precursor with a pseudosugar ring containing a 5'-ester functionality. Following appropriate functionalization of the adenine ring, the ester group was converted to the 5'-N-methylamide. The compounds, mainly 2-chloro-substituted derivatives, were tested in both binding and functional assays at human adenosine receptors (ARs), and many were found to be highly potent and selective A(3)AR agonists. Selected compounds were compared in binding to the rat A(3)AR to assess their viability for testing in rat disease models. The N(6)-(3-chlorobenzyl) and N(6)-(3-bromobenzyl) analogues displayed K(i) values at the human A(3)AR of 0.29 and 0.38 nM, respectively. Other subnanomolar affinities were observed for the following N(6) derivatives: 2,5-dichlorobenzyl, 5-iodo-2-methoxybenzyl, trans-2-phenyl-1-cyclopropyl, and 2,2-diphenylethyl. Selectivity for the human A(3)AR in comparison to the A(1)AR was the following (fold): the N(6)-(2,2-diphenylethyl) analogue 34 (1900), the N(6)-(2,5-dimethoxybenzyl) analogue 26 (1200), the N(6)-(2,5-dichlorobenzyl) and N(6)-(2-phenyl-1-cyclopropyl) analogues 20 and 33 (1000), and the N(6)-(3-substituted benzyl) analogues 17, 18, 28, and 29 (700-900). Typically, even greater selectivity ratios were obtained in comparison with the A(2A) and A(2B)ARs. The (N)-methanocarba-5'-uronamide analogues were full agonists at the A(3)AR, as indicated by the inhibition of forskolin-stimluated adenylate cyclase at a concentration of 10 microM. The N(6)-(2,2-diphenylethyl) derivative was an A(3)AR agonist in the (N)-methanocarba-5'-uronamide series, although it was an antagonist in the ribose series. Thus, many of the previously known groups that enhance A(3)AR affinity in the 9-riboside series, including those that reduce intrinsic efficacy, may be adapted to the (N)-methanocarba nucleoside series of full agonists.
Adenine↗
Sorting and activity-dependent secretion of BDNF require interaction of a specific motif with the sorting receptor carboxypeptidase e.
Activity-dependent secretion of BDNF is important in mediating synaptic plasticity, but how it is achieved is unclear. Here we uncover a sorting motif receptor-mediated mechanism for regulated secretion of BDNF. X-ray crystal structure analysis revealed a putative sorting motif, I(16)E(18)I(105)D(106), in BDNF, which when mutated at the acidic residues resulted in missorting of proBDNF to the constitutive pathway in AtT-20 cells. A V20E mutation to complete a similar motif in NGF redirected a significant proportion of it from the constitutive to the regulated pathway. Modeling and binding studies showed interaction of the acidic residues in the BDNF motif with two basic residues in the sorting receptor, carboxypeptidase E (CPE). (35)S labeling experiments demonstrated that activity-dependent secretion of BDNF from cortical neurons was obliterated in CPE knockout mice. Thus, we have identified a mechanism whereby a specific motif I(16)E(18)I(105)D(106) interacts with CPE to sort proBDNF into regulated pathway vesicles for activity-dependent secretion.
Amino Acid Motifs↗
A neoceptor approach to unraveling microscopic interactions between the human A2A adenosine receptor and its agonists.
Strategically mutated neoceptors, e.g., with anionic residues in TMs 3 and 7 intended for pairing with positively charged amine-modified nucleosides, were derived from the antiinflammatory A(2A) adenosine receptor (AR). Adenosine derivatives functionalized at the 5', 2, and N(6) positions were synthesized. The T88D mutation selectively enhanced the binding of the chain-length-optimized 5'-(2-aminoethyl)uronamide but not 5'-(2-hydroxyethyl)uronamide, suggesting a critical role of the positively charged amine. Combination of this modification with the N(6)-(2-methylbenzyl) group enhanced affinity at the Q89D- and N181D- but not the T88D-A(2A)AR. Amino groups placed near the 2- or N(6)-position only slightly affected the binding to mutant receptors. The 5'-hydrazide MRS3412 was 670- and 161-fold enhanced, in binding and functionally, respectively, at the Q89D-A(2A)AR compared to the wild-type. Thus, we identified and modeled pairs of A(2A)AR-derived neoceptor-neoligand, which are pharmacologically orthogonal with respect to the native species.
Adenosine↗
Allosteric modulation of the adenosine family of receptors.
Allosteric modulators for adenosine receptors (ARs) are of an increasing interest and may have potential therapeutic advantage over orthosteric ligands. Benzoylthiophene derivatives (including PD 81,723), 2-aminothiazolium salts, and related allosteric modulators of the A(1) AR have been studied. The benzoylthiophene derivatives were demonstrated to be selective enhancers for the A(1) AR, with little or no effect on other subtypes of ARs. Allosteric modulation of the A(2A) AR has also been reported. A(3) allosteric enhancers may be predicted to be useful against ischemic conditions. We have recently characterized two classes of A(3) AR allosteric modulators: 3-(2-pyridinyl)isoquinolines (e.g. VUF5455) and 1H-imidazo-[4,5-c]quinolin-4-amines (e.g. DU124183), which selectively decreased the agonist dissociation rate at the human A(3)AR but not at A(1) and A(2A) ARs. DU124183 left-shifted the agonist conc.-response curve for inhibition of forskolin-stimulated cAMP accumulation in intact cells expressing the human A(3)AR with up to 30% potentiation of the maximal efficacy. The increased potency of A(3) agonists was evident only in the presence of an A(3) antagonist, since VUF5455 and DU124183 also antagonized, i.e. displaced binding at the orthosteric site, with K(i) values of 1.68 and 0.82 microM, respectively. A(3)AR mutagenesis studies implicated F182(5.43) and N274(7.45) in the action of the enhancers and was interpreted using a rhodopsin-based A(3)AR molecular model, suggesting multiple binding modes. Amiloride analogues, SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5(2H)-ylidene)methanamine), and sodium ions were demonstrated to be common allosteric modulators for at least three subtypes (A(1), A(2A), and A(3)) of ARs.
Animals↗