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1.
Identification of a novel family of G protein-coupled receptor associated sorting proteins 总被引:1,自引:0,他引:1
During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein-coupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology. 相似文献
2.
《Journal of receptor and signal transduction research》2013,33(5):322-330
Although the G protein-coupled receptor (GPCR) oligomerization has been questioned during the last decade, under some premises the existence of a supramolecular organization of these receptors begins now to be widely accepted by the scientific community. Indeed, GPCR oligomers may enhance the diversity and performance by which extracellular signals are transferred to the G proteins in the process of receptor transduction, although the mechanism that underlie this phenomenon remains still unexplained. Recently, a trans-conformational switching model has been proposed as a mechanism allowing direct inhibition of receptor activation. Thus, heterotropic receptor–receptor allosteric regulations are behind the GPCR oligomeric function. Accordingly, we revise here how GPCR oligomerization impinge in several important receptor functions like biosynthesis, plasma membrane diffusion or velocity, pharmacology and signaling. Overall, the rationale of receptor oligomerization might lie in the cellular need of sensing complex extracellular signals and to translate into a simple computational mode. 相似文献
3.
The GnRH receptor is coupled to G proteins of the families Gq and G11. Gq and G11 coupling leads to intracellular signaling through the phospholipase C pathway. GnRHR coupling to other G proteins is controversial. This study provides evidence that G protein families Gs, Gi, Gq and G11 complete for binding with the GnRHR. We quantified interactions of over-expressed G proteins with GnRHR by a competitive binding approach, using measurements of second messengers, IP and cAMP. Transient co-transfection of HEK293 cells with human WT GnRHR and with stimulatory and inhibitory G proteins (Gq, G11 and Gs, Gi) led to either production or inhibition of total inositol phosphate (IP) production, depending on the G protein that was over-expressed. Studies were conducted in different human (COS7, HeLa) and rodent-derived (CHO-K1, GH3) cell lines in order to confirm that G protein promiscuity observed with the GnRHR was not limited to a particular cell type. 相似文献
4.
植物病原丝状真菌G蛋白偶联受体的研究进展 总被引:1,自引:0,他引:1
通过对丝状真菌G蛋白偶联受体(GPCR)的结构、分类以及功能方面进行综述,以期明确丝状真菌与其他模式生物GPCR之间的关系。基于已报道的模式生物及丝状真菌等不同生物中的GPCR,通过SMART保守结构域分析,以及利用Clustal X、MEGA等软件对上述GPCR进行遗传关系分析。明确丝状真菌典型GPCR具有七跨膜结构域,新型GPCR则含有PIPK、RGS等保守结构域,明确不同学者对于GPCR的分类情况,以及新型GPCR所具有的特殊功能,明确模式生物GPCR、丝状真菌GPCR分别各自聚类。丝状真菌中GPCR的数量较模式生物少,不同分类单元中真菌之间GPCR的数量也不尽相同,同时,丝状真菌GPCR除具有典型的七跨膜结构域外,还含有一些其他保守的结构域,上述研究为进一步开展其功能研究提供重要的理论基础。 相似文献
5.
The type 1 neurotensin receptor (NTS1) belongs to the G protein-coupled receptor (GPCR) family. GPCRs are involved in important physiological processes, but for many GPCRs ligand binding sites and other structural features have yet to be elucidated. Comprehensive analyses by mass spectrometry (MS) could address such issues, but they are complicated by the hydrophobic nature of the receptors. Recombinant NTS1 must be purified in the presence of detergents to maintain solubility and functionality of the receptor, to allow testing of ligand, or to allow G protein interaction. However, detergents are detrimental to MS analyses. Hence, steps need to be taken to substitute the detergents with MS-compatible polar/organic solvents. Here we report the characterization of NTS1 by electrospray ionization (ESI)-MS with emphasis on methods to transfer intact NTS1 or its proteolytic peptides into compatible solvents by protein precipitation and liquid chromatography (LC) prior to ESI-MS analyses. Molecular mass measurement of intact recombinant NTS1 was performed using a mixture of chloroform/methanol/aqueous trifluoroacetic acid as the mobile phase for size exclusion chromatography-ESI-MS analysis. In a separate experiment, NTS1 was digested with a combination of cyanogen bromide and trypsin and/or chymotrypsin. Subsequent reversed phase LC-ESI-tandem MS analysis resulted in greater than 80% sequence coverage of the NTS1 protein, including all seven transmembrane domains. This work represents the first comprehensive analysis of recombinant NTS1 using MS. 相似文献
6.
有关蛋白质功能的研究是解析生命奥秘的基础,机器学习技术在该领域已有广泛应用。利用支持向量机(support vectormachine,SVM)方法,构建一个预测蛋白质功能位点的通用平台。该平台先提取非同源蛋白质序列,再对这些序列进行特征编码(包括序列的基本信息、物化特征、结构信息及序列保守性特征等),以编码好的样本作为训练数据,利用SVM进行训练,得到敏感性、特异性、Matthew相关系数、准确率及ROC曲线等评价指标,反复测试,得到评价指标最优的SVM模型后,便可以用来预测蛋白质序列上的功能位点。该平台除了应用在预测蛋白质功能位点之外,还可以应用于疾病相关单核苷酸多态性(SNP)预测分析、预测蛋白质结构域分析、生物分子间的相互作用等。 相似文献
7.
We have identified novel G protein-coupled receptors (GPCRs) with no introns in the coding region from the human genome sequence: 322 olfactory receptors; 22 taste receptors; 128 registered GPCRs for endogenous ligands; 50 novel GPCR candidates homologous to registered GPCRs for endogenous ligands; and 59 novel GPCR candidates not homologous to registered GPCRs. The total number of GPCRs with and without introns in the human genome was estimated to be approximately 950, of which 500 are odorant or taste receptors and 450 are receptors for endogenous ligands. 相似文献
8.
Lock and chop: A novel method for the generation of a PICK1 PDZ domain and piperidine‐based inhibitor co‐crystal structure
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Douglas J. Marcotte Jean‐Christophe Hus Charles C. Banos Craig Wildes Robert Arduini Chris Bergeron Catherine A. Hession Darren P. Baker Edward Lin Kevin M. Guckian Anthone W. Dunah Laura F. Silvian 《Protein science : a publication of the Protein Society》2018,27(3):672-680
The membrane protein interacting with kinase C1 (PICK1) plays a trafficking role in the internalization of neuron receptors such as the amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA) receptor. Reduction of surface AMPA type receptors on neurons reduces synaptic communication leading to cognitive impairment in progressive neurodegenerative diseases such as Alzheimer disease. The internalization of AMPA receptors is mediated by the PDZ domain of PICK1 which binds to the GluA2 subunit of AMPA receptors and targets the receptor for internalization through endocytosis, reducing synaptic communication. We planned to block the PICK1‐GluA2 protein–protein interaction with a small molecule inhibitor to stabilize surface AMPA receptors as a therapeutic possibility for neurodegenerative diseases. Using a fluorescence polarization assay, we identified compound BIO124 as a modest inhibitor of the PICK1‐GluA2 interaction. We further tried to improve the binding affinity of BIO124 using structure‐aided drug design but were unsuccessful in producing a co‐crystal structure using previously reported crystallography methods for PICK1. Here, we present a novel method through which we generated a co‐crystal structure of the PDZ domain of PICK1 bound to BIO124. 相似文献
9.
A wide range of intracellular proteins have been demonstrated to interact with individual G protein-coupled receptors (GPCRs) and, in certain cases, to modulate their function or trafficking. However, in only a few cases have the GPCR selectivity of such interactions been investigated. Interactions between the intracellular C-terminal tails of 44 GPCRs and both neurochondrin and periplakin were assessed in pull-down studies. 23 of these interacted with neurochondrin and periplakin, 10 interacted with neither whilst nine interacted with only neurochondrin and two with only periplakin. When appropriate GIP-interacting Gq /G11 -coupled GPCRs were expressed in cells inducibly expressing neurochondrin or periplakin this resulted in a reduction in the increase in intracellular [Ca2+ ] in response to agonist. However, induction of neurochondrin or periplakin was without functional consequences for GPCRs with which they did not interact. Unlike intracellular [Ca2+ ] signals, induction of expression of either interacting protein did not inhibit agonist-mediated ERK1/2 MAPK phosphorylation. These data indicate that both periplakin and neurochondrin can interact with a wide range of GPCRs and modulate function selectively. Details of the structure of the intracellular C-terminal tail of individual receptors will be required to fully understand the basis of such selectivity. 相似文献
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We here present an improved and simplified assay to study signal transduction of the Gs class of G protein-coupled receptors (GPCRs). The assay is based on a single plasmid combining the genes for any Gs protein-coupled GPCR and the cAMP response element-related expression of enhanced yellow fluorescent protein. On transfection, stable human embryonic kidney 293 (HEK293) cell lines presented high assay sensitivity and an unprecedented signal-to-noise ratio of up to 300, even in the absence of trichostatin A. The robustness of the assay was demonstrated through the cloning of reporter gene cell lines with melanocortin 4 receptor (MC4R), the human type I pituitary adenylate cyclase-activating polypeptide receptor (hPAC1), and the two vasoactive intestinal peptide receptors (VPAC1 and VPAC2). 相似文献
12.
Konstantin E. Komolov Anshul Bhardwaj Jeffrey L. Benovic 《The Journal of biological chemistry》2015,290(34):20629-20647
G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5′-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs. 相似文献
13.
Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis. 相似文献
14.
The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ. 相似文献
15.
Clustering and synaptic targeting of PICK1 requires direct interaction between the PDZ domain and lipid membranes 总被引:1,自引:0,他引:1
Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ domain can directly interact with lipid membranes. The PDZ domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved 'Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ domain, but also provided direct evidence supporting the functional relevance of the PDZ-lipid interaction. 相似文献
16.
Dominique Massotte 《生物化学与生物物理学报:生物膜》2003,1610(1):77-89
G protein-coupled receptors, whose topology shows seven transmembrane domains, form the largest known family of receptors involved in higher organism signal transduction. These receptors are generally of low natural abundance and overexpression is usually a prerequisite to their structural or functional characterisation. The baculovirus-insect cell system constitutes a versatile tool for the maximal production of receptors. This heterologous expression system also provides interesting alternatives for receptor functional studies in a well-controlled cellular context. 相似文献
17.
The higher-order structure of G protein-coupled receptors (GPCRs) in membranes may involve dimerization and formation of even larger oligomeric complexes. Here, we have investigated the organization of the prototypical GPCR rhodopsin in its native membrane by electron and atomic force microscopy (AFM). Disc membranes from mice were isolated and observed by AFM at room temperature. In all experimental conditions, rhodopsin forms structural dimers organized in paracrystalline arrays. A semi-empirical molecular model for the rhodopsin paracrystal is presented validating our previously reported results. Finally, we compare our model with other currently available models describing the supramolecular structure of GPCRs in the membrane. 相似文献
18.
Heather E. McFarlane Daniela Mutwil-Anderwald Jana Verbančič Kelsey L. Picard Timothy E. Gookin Anja Froehlich David Chakravorty Luisa M. Trindade Jose M. Alonso Sarah M. Assmann Staffan Persson 《Developmental cell》2021,56(10):1484-1497.e7
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G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins which conduct a wide range of biological roles and represent significant drug targets. Most biophysical and structural studies of GPCRs have been conducted on detergent-solubilised receptors, and it is clear that detergents can have detrimental effects on GPCR function. Simultaneously, there is increasing appreciation of roles for specific lipids in modulation of GPCR function. Lipid nanoparticles such as nanodiscs and styrene maleic acid lipid particles (SMALPs) offer opportunities to study integral membrane proteins in lipid environments, in a form that is soluble and amenable to structural and biophysical experiments. Here, we review the application of lipid nanoparticle technologies to the study of GPCRs, assessing the relative merits and limitations of each system. We highlight how these technologies can provide superior platforms to detergents for structural and biophysical studies of GPCRs and inform on roles for protein-lipid interactions in GPCR function. 相似文献