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2014, PloS one
The activation of the G-protein transducin (Gt) by rhodopsin (Rho) has been intensively studied for several decades. It is the best understood example of GPCR activation mechanism and serves as a template for other GPCRs. The structure of the Rho/G protein complex, which is transiently formed during the signaling reaction, is of particular interest. It can help understanding the molecular details of how retinal isomerization leads to the G protein activation, as well as shed some light on how GPCR recognizes its cognate G protein. The native Rho/Gt complex isolated from bovine retina suffers from low stability and loss of the retinal ligand. Recently, we reported that constitutively active mutant of rhodopsin E113Q forms a Rho/Gt complex that is stable in detergent solution. Here, we introduce methods for a large scale preparation of the complex formed by the thermo-stabilized and constitutively active rhodopsin mutant N2C/M257Y/D282C(RhoM257Y) and the native Gt purified from bovine...
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2009, Biophysical Journal
2011, Biochemistry
2012, The FASEB Journal
2014, Nature communications
G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the 'finger loop' region (ArrFL1-4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interfa...
2015, Frontiers in Pharmacology
2009, Biochemistry
2013, Journal of Structural Biology
2000
G protein-coupled receptors (GPCRs) are seven transmem- brane domain proteins that transduce extracellular signals across the plasma membrane and couple to the heterotrimeric family of G proteins. Like most intrinsic membrane proteins, GPCRs are capable of oligomerization, the function of which has only been established for a few different receptor systems. One challenge in understanding the function of oligomers relates
2004, Journal of Biological Chemistry
2013, Current Opinion in Structural Biology
2013, Biomedical spectroscopy and imaging
G protein-coupled receptors (GPCR) are integral membrane proteins that transmit signals from external stimuli to the cell interior via activation of GTP-binding proteins (G proteins) thereby mediating key sensorial, hormonal, metabolic, immunological, and neurotransmission processes. Elucidating their structure and mechanism of interaction with extracellular and intracellular binding partners is of fundamental importance and highly relevant to rational design of new effective drugs. Surface plasmon resonance (SPR) has become a method of choice for studying biomolecular interactions at interfaces because measurements take place in real-time and do not require labeling of any of the interactants. However, due to the particular challenges imposed by the high hydrophobicity of membrane proteins and the great diversity of receptor-stimulating ligands, the application of this technique to characterize interactions of GPCR is still in the developmental phase. Here we give an overview of th...
2008, Journal of Molecular Biology
2010, Biochemical Journal
2008, Journal of Biological Chemistry
2013, Methods in enzymology
Constituting the largest group of membrane proteins identified in the human genome, G protein-coupled receptors (GPCRs) help control many physiological processes by responding to various stimuli. As targets for more than 40% of all prescribed pharmaceuticals, detailed understanding of GPCR structures is vital for the design and development of more specific medications and improved patient therapies. But structural information for membrane proteins and GPCRs, in particular, is limited despite considerable interest. The major impediment to obtaining sufficient quantities of highly purified GPCRs in their native form for crystallization lies in their low tissue levels, poor yields, and stability. The only exception is rhodopsin, which is abundantly expressed in the eye and stabilized by its covalently bound chromophore, 11-cis-retinal. Expression systems and purification protocols have yet to be developed for all other GPCRs. Here, we present a novel expression system for human GPCRs i...
2008, Biochemistry
2012, Proceedings of the National Academy of Sciences
2011, Cellular and Molecular Life Sciences
1998, Human Molecular Genetics
2012, PLoS ONE
2014, The Journal of biological chemistry
Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking....
2010, The Faseb Journal
2003, Annual Review of Physiology
2011, Journal of Structural Biology
2007, Developmental Neurobiology
2004, Journal of Biological Chemistry
2014, Biochimie
2007, Biochemistry
2005, Current Protein & Peptide Science
2007, Journal of Biological Chemistry
The cytoplasmic sides of transmembrane helices 3 and 6 of G-protein-coupled receptors are connected by a network of ionic interactions that play an important role in maintaining its inactive conformation. To investigate the role of such a network in rhodopsin structure and function, we have constructed single mutants at position 134 in helix 3 and at positions 247 and 251 in helix 6, as well as combinations of these to obtain double mutants involving the two helices. These mutants have been expressed in COS-1 cells, immunopurified using the rho-1D4 antibody, and studied by UV-visible spectrophotometry. Most of the single mutations did not affect chromophore formation, but double mutants, especially those involving the T251K mutant, resulted in low yield of protein and impaired 11-cis-retinal binding. Single mutants E134Q, E247Q, and E247A showed the ability to activate transducin in the dark, and E134Q and E247A enhanced activation upon illumination, with regard to wild-type rhodopsin. Mutations E247A and T251A (in E134Q/E247A and E134Q/T251A double mutants) resulted in enhanced activation compared with the single E134Q mutant in the dark. A role for Thr(251) in this network is proposed for the first time in rhodopsin. As a result of these mutations, alterations in the hydrogen bond interactions between the amino acid side chains at the cytoplasmic region of transmembrane helices 3 and 6 have been observed using molecular dynamics simulations. Our combined experimental and modeling results provide new insights into the details of the structural determinants of the conformational change ensuing photoactivation of rhodopsin.
2004, Journal of Biological Chemistry
2014, PLoS ONE
2004, Journal of Biological Chemistry
2011, Biochemistry
PLoS ONE
Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1•GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] b...
2010, The EMBO journal
Functional asymmetry of G-protein-coupled receptor (GPCR) dimers has been reported for an increasing number of cases, but the molecular architecture of signalling units associated to these dimers remains unclear. Here, we characterized the molecular complex of the melatonin MT₁ receptor, which directly and constitutively couples to G(i) proteins and the regulator of G-protein signalling (RGS) 20. The molecular organization of the ternary MT₁/G(i)/RGS20 complex was monitored in its basal and activated state by bioluminescence resonance energy transfer between probes inserted at multiple sites of the complex. On the basis of the reported crystal structures of G(i) and the RGS domain, we propose a model wherein one G(i) and one RGS20 protein bind to separate protomers of MT₁ dimers in a pre-associated complex that rearranges upon agonist activation. This model was further validated with MT₁/MT₂ heterodimers. Collectively, our data extend the concept of asymmetry within GPCR dimers, rei...
2011, Progress in Lipid Research
2013, The Journal of Biological Chemistry
2003, Proceedings of the National Academy of Sciences
2010, Parasitology Research
2012, PLoS Pathogens
2004, Biochemistry
2012, The FASEB Journal
1997, Journal of Biological Chemistry
2009, Proceedings of The National Academy of Sciences
2011, Journal of Biological Chemistry
2000, Biochemistry