共查询到20条相似文献，搜索用时 46 毫秒
1.
Rosenbaum DM Cherezov V Hanson MA Rasmussen SG Thian FS Kobilka TS Choi HJ Yao XJ Weis WI Stevens RC Kobilka BK 《Science (New York, N.Y.)》2007,318(5854):1266-1273
The beta2-adrenergic receptor (beta2AR) is a well-studied prototype for heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the beta2AR and to facilitate its crystallization, we engineered a beta2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR ("beta2AR-T4L") and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of beta2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins. 相似文献
2.
Rhodopsin mutants that bind but fail to activate transducin 总被引：19，自引：0，他引：19
R R Franke B K?nig T P Sakmar H G Khorana K P Hofmann 《Science (New York, N.Y.)》1990,250(4977):123-125
Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt. 相似文献
3.
J L Benovic A DeBlasi W C Stone M G Caron R J Lefkowitz 《Science (New York, N.Y.)》1989,246(4927):235-240
The beta-adrenergic receptor kinase (beta-ARK), which specifically phosphorylates only the agonist-occupied form of the beta-adrenergic and closely related receptors, appears to be important in mediating rapid agonist-specific (homologous) desensitization. The structure of this enzyme was elucidated by isolating clones from a bovine brain complementary DNA library through the use of oligonucleotide probes derived from partial amino acid sequence. The beta-ARK cDNA codes for a protein of 689 amino acids (79.7 kilodaltons) with a protein kinase catalytic domain that bears greatest sequence similarity to protein kinase C and the cyclic adenosine monophosphate (cyclic AMP)--dependent protein kinase. When this clone was inserted into a mammalian expression vector and transfected into COS-7 cells, a protein that specifically phosphorylated the agonist-occupied form of the beta 2-adrenergic receptor and phosphorylated, much more weakly, the light-bleached form of rhodopsin was expressed. RNA blot analysis revealed a messenger RNA of four kilobases with highest amounts in brain and spleen. Genomic DNA blot analysis also suggests that beta-ARK may be the first sequenced member of a multigene family of receptor kinases. 相似文献
4.
Jaakola VP Griffith MT Hanson MA Cherezov V Chien EY Lane JR Ijzerman AP Stevens RC 《Science (New York, N.Y.)》2008,322(5905):1211-1217
The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors. 相似文献
5.
Chimeric alpha 2-,beta 2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity 总被引：47，自引：0，他引：47
B K Kobilka T S Kobilka K Daniel J W Regan M G Caron R J Lefkowitz 《Science (New York, N.Y.)》1988,240(4857):1310-1316
The alpha 2 and beta 2 adrenergic receptors, both of which are activated by epinephrine, but which can be differentiated by selective drugs, have opposite effects (inhibitory and stimulatory) on the adenylyl cyclase system. The two receptors are homologous with each other, rhodopsin, and other receptors coupled to guanine nucleotide regulatory proteins and they contain seven hydrophobic domains, which may represent transmembrane spanning segments. The function of specific structural domains of these receptors was determined after construction and expression of a series of chimeric alpha 2-,beta 2-adrenergic receptor genes. The specificity for coupling to the stimulatory guanine nucleotide regulatory protein lies within a region extending from the amino terminus of the fifth hydrophobic domain to the carboxyl terminus of the sixth. Major determinants of alpha 2- and beta 2-adrenergic receptor agonist and antagonist ligand binding specificity are contained within the seventh membrane spanning domain. Chimeric receptors should prove useful for elucidating the structural basis of receptor function. 相似文献
6.
Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR. 相似文献
7.
Xu F Wu H Katritch V Han GW Jacobson KA Gao ZG Cherezov V Stevens RC 《Science (New York, N.Y.)》2011,332(6027):322-327
Activation of G protein-coupled receptors upon agonist binding is a critical step in the signaling cascade for this family of cell surface proteins. We report the crystal structure of the A(2A) adenosine receptor (A(2A)AR) bound to an agonist UK-432097 at 2.7 angstrom resolution. Relative to inactive, antagonist-bound A(2A)AR, the agonist-bound structure displays an outward tilt and rotation of the cytoplasmic half of helix VI, a movement of helix V, and an axial shift of helix III, resembling the changes associated with the active-state opsin structure. Additionally, a seesaw movement of helix VII and a shift of extracellular loop 3 are likely specific to A(2A)AR and its ligand. The results define the molecule UK-432097 as a "conformationally selective agonist" capable of receptor stabilization in a specific active-state configuration. 相似文献
8.
Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit 总被引：6，自引：0，他引：6
K King H G Dohlman J Thorner M G Caron R J Lefkowitz 《Science (New York, N.Y.)》1990,250(4977):121-123
To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors. 相似文献
9.
Cloning, sequencing, and expression of the gene coding for the human platelet alpha 2-adrenergic receptor 总被引：55，自引：0，他引：55
B K Kobilka H Matsui T S Kobilka T L Yang-Feng U Francke M G Caron R J Lefkowitz J W Regan 《Science (New York, N.Y.)》1987,238(4827):650-656
The gene for the human platelet alpha 2-adrenergic receptor has been cloned with oligonucleotides corresponding to the partial amino acid sequence of the purified receptor. The identity of this gene has been confirmed by the binding of alpha 2-adrenergic ligands to the cloned receptor expressed in Xenopus laevis oocytes. The deduced amino acid sequence is most similar to the recently cloned human beta 2- and beta 1-adrenergic receptors; however, similarities to the muscarinic cholinergic receptors are also evident. Two related genes have been identified by low stringency Southern blot analysis. These genes may represent additional alpha 2-adrenergic receptor subtypes. 相似文献
10.
Harrison T Samuel BU Akompong T Hamm H Mohandas N Lomasney JW Haldar K 《Science (New York, N.Y.)》2003,301(5640):1734-1736
Erythrocytic mechanisms involved in malarial infection are poorly understood. We have found that signaling via the erythrocyte beta2-adrenergic receptor and heterotrimeric guanine nucleotide-binding protein (Galphas) regulated the entry of the human malaria parasite Plasmodium falciparum. Agonists that stimulate cyclic adenosine 3',5'-monophosphate production led to an increase in malarial infection that could be blocked by specific receptor antagonists. Moreover, peptides designed to inhibit Galphas protein function reduced parasitemia in P. falciparum cultures in vitro, and beta-antagonists reduced parasitemia of P. berghei infections in an in vivo mouse model. Thus, signaling via the erythrocyte beta2-adrenergic receptor and Galphas may regulate malarial infection across parasite species. 相似文献
11.
Three-dimensional structure of an oncogene protein: catalytic domain of human c-H-ras p21 总被引：60，自引：0，他引：60
A M de Vos L Tong M V Milburn P M Matias J Jancarik S Noguchi S Nishimura K Miura E Ohtsuka S H Kim 《Science (New York, N.Y.)》1988,239(4842):888-893
The crystal structure at 2.7 A resolution of the normal human c-H-ras oncogene protein lacking a flexible carboxyl-terminal 18 residue reveals that the protein consists of a six-stranded beta sheet, four alpha helices, and nine connecting loops. Four loops are involved in interactions with bound guanosine diphosphate: one with the phosphates, another with the ribose, and two with the guanine base. Most of the transforming proteins (in vivo and in vitro) have single amino acid substitutions at one of a few key positions in three of these four loops plus one additional loop. The biological functions of the remaining five loops and other exposed regions are at present unknown. However, one loop corresponds to the binding site for a neutralizing monoclonal antibody and another to a putative "effector region"; mutations in the latter region do not alter guanine nucleotide binding or guanosine triphosphatase activity but they do reduce the transforming activity of activated proteins. The data provide a structural basis for understanding the known biochemical properties of normal as well as activated ras oncogene proteins and indicate additional regions in the molecule that may possibly participate in other cellular functions. 相似文献
12.
Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp) 总被引：65，自引：0，他引：65
M Ruff S Krishnaswamy M Boeglin A Poterszman A Mitschler A Podjarny B Rees J C Thierry D Moras 《Science (New York, N.Y.)》1991,252(5013):1682-1689
The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence. 相似文献
13.
Ahumada A Slusarski DC Liu X Moon RT Malbon CC Wang HY 《Science (New York, N.Y.)》2002,298(5600):2006-2010
The Frizzled-2 receptor (Rfz2) from rat binds Wnt proteins and can signal by activating calcium release from intracellular stores. We show that wild-type Rfz2 and a chimeric receptor consisting of the extracellular and transmembrane portions of the beta2-adrenergic receptor with cytoplasmic domains of Rfz2 also signaled through modulation of cyclic guanosine 3',5'-monophosphate (cGMP). Activation of either receptor led to a decline in the intracellular concentration of cGMP, a process that was inhibited in cells treated with pertussis toxin, reduced by suppression of the expression of the heterotrimeric GTP-binding protein (G protein) transducin, and suppressed through inhibition of cGMP-specific phosphodiesterase (PDE) activity. Moreover, PDE inhibitors blocked Rfz2-induced calcium transients in zebrafish embryos. Thus, Frizzled-2 appears to couple to PDEs and calcium transients through G proteins. 相似文献
14.
Members of the seven transmembrane receptor superfamily bind a remarkable variety of ligands, from neurotransmitters to odorants, and activate a spectacular array of G protein signaling molecules. These G-protein coupled receptors (GPCRs) are important in many cellular functions and so there has been great interest in elucidating how they transmit their signals to the interior of the cell after activation by ligand. As Bourne and Meng explain in their Perspective, the molecular movements of activated GPCRs are becoming clear now that the first crystal structure of a GPCR (rhodopsin, the light-trapping receptor found in the retina of the eye) has been reported (Palczweski et al.). 相似文献
15.
B A Jameson P E Rao L I Kong B H Hahn G M Shaw L E Hood S B Kent 《Science (New York, N.Y.)》1988,240(4857):1335-1339
The human immunodeficiency virus type 1 (HIV-1) uses the CD4 protein as a receptor for infection of susceptible cells. A candidate structure for the HIV-1 binding site on the CD4 protein was identified by epitope mapping with a family of eight functionally distinct CD4-specific monoclonal antibodies in conjunction with a panel of large CD4-derived synthetic peptides. All of the seven epitopes that were located reside within two immunoglobulin-like disulfide loops situated between residues 1 and 168 of the CD4 protein. The CD4-specific monoclonal antibody OKT4A, a potent inhibitor of HIV-1 binding, recognized a site between residues 32 and 47 on the CD4 protein. By analogy to other members of the immunoglobulin superfamily of proteins, this particular region has been predicted to exist as a protruding loop. A synthetic analog of this loop (residues 25 to 58) showed a concentration-dependent inhibition of HIV-1-induced cell fusion. It is proposed that a loop extending from residues 37 to 53 of the CD4 protein is a binding site for the AIDS virus. 相似文献
16.
Chien EY Liu W Zhao Q Katritch V Han GW Hanson MA Shi L Newman AH Javitch JA Cherezov V Stevens RC 《Science (New York, N.Y.)》2010,330(6007):1091-1095
Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications. 相似文献
17.
Transducin activation by rhodopsin without a covalent bond to the 11-cis-retinal chromophore 总被引：3，自引：0，他引：3
Rhodopsin and the visual pigments are a distinct group within the family of G-protein-linked receptors in that they have a covalently bound ligand, the 11-cis-retinal chromophore, whereas all of the other receptors bind their agonists through noncovalent interactions. The retinal chromophore in rhodopsin is bound by means of a protonated Schiff base linkage to the epsilon-amino group of Lys-296. Two rhodopsin mutants have been constructed, K296G and K296A, in which the covalent linkage to the chromophore is removed. Both mutants form a pigment with an absorption spectrum close to that of the wild type when reconstituted with the Schiff base of an n-alkylamine and 11-cis-retinal. In addition, the pigment formed from K296G and the n-propylamine Schiff base of 11-cis-retinal was found to activate transducin in a light-dependent manner, with 30 to 40% of the specific activity measured for the wild-type protein. It appears that the covalent bond is not essential for binding of the chromophore or for catalytic activation of transducin. 相似文献
18.
Perry SJ Baillie GS Kohout TA McPhee I Magiera MM Ang KL Miller WE McLean AJ Conti M Houslay MD Lefkowitz RJ 《Science (New York, N.Y.)》2002,298(5594):834-836
Catecholamines signal through the beta2-adrenergic receptor by promoting production of the second messenger adenosine 3',5'-monophosphate (cAMP). The magnitude of this signal is restricted by desensitization of the receptors through their binding to beta-arrestins and by cAMP degradation by phosphodiesterase (PDE) enzymes. We show that beta-arrestins coordinate both processes by recruiting PDEs to activated beta2-adrenergic receptors in the plasma membrane of mammalian cells. In doing so, the beta-arrestins limit activation of membrane-associated cAMP-activated protein kinase by simultaneously slowing the rate of cAMP production through receptor desensitization and increasing the rate of its degradation at the membrane. 相似文献
19.
Signaling by heterotrimeric GTP-binding proteins (G proteins) drives numerous cellular processes. The number of G protein molecules activated by a single membrane receptor is a determinant of signal amplification, although in most cases this parameter remains unknown. In retinal rod photoreceptors, a long-lived photoisomerized rhodopsin molecule activates many G protein molecules (transducins), yielding substantial amplification and a large elementary (single-photon) response, before rhodopsin activity is terminated. Here we report that the elementary response in olfactory transduction is extremely small. A ligand-bound odorant receptor has a low probability of activating even one G protein molecule because the odorant dwell-time is very brief. Thus, signal amplification in olfactory transduction appears fundamentally different from that of phototransduction. 相似文献
20.
Although signals controlled by single molecules are expected to be inherently variable, rod photoreceptors generate reproducible responses to single absorbed photons. We show that this unexpected reproducibility-the consistency of amplitude and duration of rhodopsin activity-varies in a graded and systematic manner with the number but not the identity of phosphorylation sites on rhodopsin's C terminus. These results indicate that each phosphorylation site provides an independent step in rhodopsin deactivation and that collectively these steps tightly control rhodopsin's active lifetime. Other G protein cascades may exploit a similar mechanism to encode accurately the timing and number of receptor activation. 相似文献