Constitutively active rhodopsin mutants causing night blindness are effectively phosphorylated by GRKs but differ in arrestin-1 binding

Vishnivetskiy, Sergey A.; Ostermaier, Martin K.; Singhal, Ankita; Panneels, Valérie; Homan, Kristoff T.; Glukhova, Alisa; Sligar, Stephen G.; Tesmer, John J.G.; Schertler, Gebhard F. X.; Standfuss, Joerg; Gurevich, Vsevolod V.

doi:10.1016/j.cellsig.2013.07.009

Cited by 30 publications

(25 citation statements)

References 92 publications

(129 reference statements)

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“…This conclusion is supported by recent studies using rhodopsin-embedded nanodiscs, which have shown that one Rho* is sufficient for coupling to and activating G T (33)(34). Moreover, based on nanodisc reconstitution, monomeric rhodopsin has been shown to be sufficient for phosphorylation by G proteincoupled receptor kinase and interaction with arrestin (35)(36). The recent X-ray structure of a rhodopsin-arrestin complex (37) further illustrated that rhodopsin binds arrestin in a 1:1 stoichiometry.…”

Section: Structure-function Studies Of Rhodopsin-g Protein Signalingsupporting

confidence: 60%

Isolation and structure–function characterization of a signaling-active rhodopsin–G protein complex

Gao¹,

Westfield²,

Erickson³

et al. 2017

Journal of Biological Chemistry

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The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (G T ). This results in the dissociation of G T into its component ␣ T -GTP and ␤ 1 ␥ 1 subunit complex. Structural information for the Rho*-G T complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (G T *) comprising a G␣ T /G␣ i1 chimera (␣ T *) and ␤ 1 ␥ 1 . The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to G␣ T * is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one G T *. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the ␤ 2 -adrenergic receptor-G S complex, including a flexible ␣ T * helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a highresolution structure of this important signaling complex.G protein-coupled receptors (GPCRs), 3 the largest family of transmembrane proteins, are the targets for nearly 50% of all pharmaceutical drugs (1). These receptors modulate cellular responses to a vast array of extracellular signals through the activation of heterotrimeric G proteins, with the active state being defined as the complex that forms between the agonistbound (or light-stimulated) GPCR and nucleotide-free G protein (2). Attempts to obtain structural information for GPCRs, and especially for signaling-active GPCR-G protein complexes, have garnered a great deal of interest, both as a means to better understand the underlying mechanisms by which this important family of receptors mediates a wide range of biological outcomes and as a critical step in the design of more selective and effective drug treatments. Recent technological advancements, including novel protein engineering (3), in meso crystallization (4), and micro-focus beamlines at synchrotron facilities (5), have ushered in significant progress in the determination of high-resolution GPCR structures. However, only a few of those structures are of activated GPCRs, and thus far, only two GPCR-G protein complex structures have been solved, namely that of the ␤ 2 -adrenergic receptor-G S protein complex (6) and the calcitonin receptor-G S protein complex (7). Furthermore, virtually all of these structures, with the exception of rhodopsin, have been obtained with G...

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Section: Structure-function Studies Of Rhodopsin-g Protein Signalingsupporting

confidence: 60%

Isolation and structure–function characterization of a signaling-active rhodopsin–G protein complex

Gao¹,

Westfield²,

Erickson³

et al. 2017

Journal of Biological Chemistry

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“…4, Right Middle Inset), whose relative position has been implicated in arrestin-biased agonism in β-adrenergic receptors (36) and arrestin binding to rhodopsin (37). Also, the introduction of a salt-bridge between TM3 and TM7 by the night blindness causing rhodopsin mutant G90D reduces its ability to bind arrestin (38,39). Together, the finger and lariat loop could thus create an activity sensor that specifically interacts with helices critical for GPCR activation.…”

Section: Resultsmentioning

confidence: 99%

Functional map of arrestin-1 at single amino acid resolution

Ostermaier

Peterhans

Jaussi

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Arrestins function as adapter proteins that mediate G proteincoupled receptor (GPCR) desensitization, internalization, and additional rounds of signaling. Here we have compared binding of the GPCR rhodopsin to 403 mutants of arrestin-1 covering its complete sequence. This comprehensive and unbiased mutagenesis approach provides a functional dimension to the crystal structures of inactive, preactivated p44 and phosphopeptide-bound arrestins and will guide our understanding of arrestin-GPCR complexes. The presented functional map quantitatively connects critical interactions in the polar core and along the C tail of arrestin. A series of amino acids (Phe375, Phe377, Phe380, and Arg382) anchor the C tail in a position that blocks binding of the receptor. Interaction of phosphates in the rhodopsin C terminus with Arg29 controls a C-tail exchange mechanism in which the C tail of arrestin is released and exposes several charged amino acids (Lys14, Lys15, Arg18, Lys20, Lys110, and Lys300) for binding of the phosphorylated receptor C terminus. In addition to this arrestin phosphosensor, our data reveal several patches of amino acids in the finger (Gln69 and Asp73-Met75) and the lariat loops (L249-S252 and Y254) that can act as direct binding interfaces. A stretch of amino acids at the edge of the C domain (Trp194-Ser199, Gly337-Gly340, Thr343, and Thr345) could act as membrane anchor, binding interface for a second rhodopsin, or rearrange closer to the central loops upon complex formation. We discuss these interfaces in the context of experimentally guided docking between the crystal structures of arrestin and light-activated rhodopsin.visual system | scanning mutagenesis | membrane receptor | cell signaling | protein engineering T he human genome encodes more than 800 G protein-coupled receptors (GPCRs), which mediate signaling between cells and provide an important link to our environment as the principal receptors for taste, smell, and vision. The visual system with its photoreceptor rhodopsin is an excellent system to understand GPCR signaling, as detailed information exists on the structures and dynamic interactions of the protein constituents (1). G protein-mediated signaling by light-activated rhodopsin is terminated by a process that begins with the phosphorylation of rhodopsin's C terminus by the rhodopsin kinase GRK1. The phosphorylated, light-activated rhodopsin binds then to arrestin-1, which stops signaling by occluding the G protein-binding site. Further cloning efforts yielded two ubiquitously expressed nonvisual arrestins (arrestin-2 and arrestin-3 or β-arrestin-1 and β-arrestin-2) and the cone-specific arrestin-4. It seems clear today that most GPCRs share a common mechanism of signal termination involving receptor phosphorylation and the binding of arrestins. Arrestin-bound receptors may be internalized and degraded, internalized and recycled, and/or initiate G proteinindependent signaling (2).In recent years there has been tremendous progress in the structure determination of active GPCR states includ...

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“…Principally such distinct conformations could control binding of arrestins either directly or more indirectly by favoring distinct phosphorylation patterns added by different GRKs. GRK1 and GRK2, for example, are able to promote high affinity binding of arrestin-1 to R*-P, whereas GRK5 was much less efficient [44 ]. Similarly, GRK2 and GRK3 effectively recruited arrestin-3 (also known as b-arrestin 2) for desensitization of vasopressin receptor V2, while GRK5 and GRK6 had much less influence on arrestin-3 recruitment [45].…”

Section: Phosphorylation-dependent Activation Of Arrestinmentioning

confidence: 97%