Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

Russwurm, Michael; Müllershausen, Florian; Friebe, Andreas; Jäger, Roland; Russwurm, Corina; Koesling, Doris

doi:10.1042/bj20070348

Cited by 127 publications

(154 citation statements)

References 44 publications

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“…Furthermore, due to its high affinity and selectivity, PKG II CNB-B is a good candidate as a scaffold for the generation of FRET-based protein biosensors, which could be used to measure the spatiotemporal dynamics of NO-cGMP signaling in cells. Historically, cGMPresponsive FRET biosensors have been constructed using the PKG I CNBs (33)(34)(35)(36). Based on our previous work examining PKG I CNBs (4, 17), we can explain different cGMP selectivity profiles seen in these sensors (37).…”

Section: Discussionmentioning

confidence: 99%

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

Campbell¹,

Kim

et al. 2016

Journal of Biological Chemistry

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Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the ␣C-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.

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Section: Discussionmentioning

confidence: 99%

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

Campbell¹,

Kim

et al. 2016

Journal of Biological Chemistry

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“…This construct contains only the cGMP-binding sites, which are identical in cGKI␣ and cGKI␤ but lacks the N-terminal region that differs between the isozymes. Binding of the agonist cGMP to the FRET construct induces a conformational change that can be monitored as altered FRET signal (30), whereas binding of an antagonist does not produce a FRET change. In line with a partial agonistic activity of Rp-PET, increasing concentrations of Rp-PET caused a similar, albeit weaker, FRET change as the known agonists cGMP and PET (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For these measurements, the two cGMP-binding sites of bovine cGKI were sandwiched between CFP and YFP, respectively (30). This construct contains only the cGMP-binding sites, which are identical in cGKI␣ and cGKI␤ but lacks the N-terminal region that differs between the isozymes.…”

Section: Resultsmentioning

confidence: 99%

The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

Valtcheva

Nestorov

Beck

et al. 2009

Journal of Biological Chemistry

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cGMP is a cyclic-nucleotide second messenger with multiple targets and functions. Small-molecule modulators of cGMP generators and effectors are important biochemical tools as well as established and prospective drugs for the treatment of human diseases, such as erectile dysfunction, pulmonary hypertension, and various cardiovascular disorders (1-3). cGMP is generated by nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and can bind to and modulate the activity of at least three classes of cGMP effector proteins: cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs), 2 cyclic nucleotide-gated cation channels, and cGMP-dependent protein kinases (cGKs, also known as protein kinase G or PKG) (4).Based on pharmacological and genetic studies, the cGK type I (cGKI) is considered the major mediator of cGMP signaling in many tissues including the cardiovascular system (5-8). The mammalian cGKI is a cytosolic Ser/Thr protein kinase comprising an N-terminal regulatory domain with two cGMPbinding sites and a C-terminal catalytic domain. It exists in two isoforms termed cGKI␣ and cGKI␤. The isozymes have identical cGMP-binding sites and catalytic domains but differ in their N-terminal ϳ100 amino acids, which contribute to homodimerization, sensitivity to cGMP activation, and interaction with anchoring and substrate proteins. Recent in vivo studies with transgenic mice demonstrated that both isoforms can induce smooth muscle relaxation and vasodilation (9), but the respective molecular mechanisms behind these effects are controversial (6, 10). Similarly, opposing effects of cGMP/cGKI signaling have been reported on the growth and phenotype of vascular smooth muscle cells (VSMCs) (11,12). The inconsistency of the results concerning the function of cGKI might in part be related to unexpected effects of the pharmacological cGKI activators and inhibitors that are commonly used to distinguish between cGKI-dependent and cGKI-independent signaling. For instance, cGMP analogues can bind to multiple * This work was supported by grants from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 The abbreviations used are: PDE, phosphodiesterase; cGKI, cGMP-dependent protein kinase type I; PET, 8-Br-PET-cGMP; Rp-PET, Rp-8-Br-PETcGMPS; 8-Br-PET-cGMP, ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphate; Rp-8-pCPT-cGMPS, 8-(4-chlorophenylthio)-guanosine-3Ј, 5Ј-cyclic monophosphorothioate, Rp-isomer; Rp-8-Br-PET-cGMPS, ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphorothioate, Rp-isomer; VASP, vasodilator-stimulated phosphoprotein; VSMC, vascular smooth muscle cell; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ESI-MS, electrospray ionization-mass spectrometry; ...

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“…In previous work we used the genetically encoded cGMP sensor cGi500 (20) to visualize cGMP dynamics in the PQR O 2 -sensing neuron (14). We showed that a rise in O 2 stimulates a tonic rise in cGMP that requires the GCY-35 soluble guanylate cyclase and that the Ca 2+ influx resulting from gating of cGMP channels feeds back to limit O 2 -evoked rises in cGMP by stimulating cGMP hydrolysis (14) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Modulation of sensory information processing by a neuroglobin in Caenorhabditis elegans

Oda

Toyoshima

Bono

2017

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

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T he response properties of sensory neurons can be characterized by tuning curves that relate stimulus parameters to the evoked response (1, 2). Some sensory neurons show dynamic ranges that span several orders of stimulus magnitude (e.g., odor concentration), whereas others show remarkably narrow tuning curves. For example, glomus cells of the carotid body show oxygenevoked responses that are tuned to a twofold to threefold change in O 2 levels at the physiologically appropriate O 2 concentration range (3, 4). How such sharp tuning is achieved is poorly understood.Neuroglobins are members of the globin family of hemebinding proteins expressed mainly in neurons (5). They have been described throughout metazoa, from cnidarians to man (6). Their physiological functions are unclear. They have been proposed to metabolize reactive oxygen species (ROS), signal redox state, store O 2 , control apoptosis, and negatively regulate Gi/o signaling (7). The genome of Caenorhabditis elegans encodes an unusually large family of globins, and many are expressed in neurons (8). One of these, GLOBIN-5 (GLB-5), is expressed in the oxygen sensing neurons URX, AQR, PQR, and BAG, where it accumulates at dendritic endings (9, 10). C. elegans avoids both normoxia (21% O 2 ) and hypoxia (11). Avoidance of 21% O 2 enables the animal to escape the surface and is mediated by O 2 receptors, most importantly the glb-5-expressing URX, AQR, and PQR neurons (12). Like vertebrate neuroglobins, GLB-5 has the spectroscopic fingerprints of a hexa-coordinated heme iron and rapidly oxidizes to the ferric state in normoxia (9). The glb-5 gene is defective in the domesticated reference strain of C. elegans, N2 (Bristol), but natural isolates encode a functional allele, glb-5(Haw) (9, 10) (Haw refers to Hawaii, the geographical origin of the natural isolate in which this allele was first described). Behavioral and Ca 2+ imaging studies suggest that functional GLB-5 alters the properties of the O 2 receptors and C. elegans' O 2 responses (9, 10). However, how GLB-5 alters the representation of environmental information in these neurons leading to behavioral change is unknown.The URX O 2 receptors exhibit phasic-tonic signaling properties and, in response to changes in O 2 concentration, evoke both transient behavioral responses that are coupled to the rate of change of O 2 , dO 2 /dt, and more persistent behavioral responses coupled to O 2 levels (8, 13). The transient responses are reversals and turns that allow C. elegans to navigate O 2 gradients. The sustained responses involve persistent changes in the rate of movement that enable feeding animals to escape 21% O 2 or to accumulate in preferred lower O 2 environments. Besides GLB-5, the URX neurons express another putative molecular O 2 sensor, a soluble guanylate cyclase composed of GCY-35 and GCY-36 (guanylate cyclase) subunits (11,13,14). These soluble guanylate cyclases have a heme-nitric oxide/oxygen (H-NOX) binding domain that appears to stimulate cGMP production upon binding molecular O 2 (10, ...

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Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

Cited by 127 publications

References 44 publications

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

The Commonly Used cGMP-dependent Protein Kinase Type I (cGKI) Inhibitor Rp-8-Br-PET-cGMPS Can Activate cGKI in Vitro and in Intact Cells

Modulation of sensory information processing by a neuroglobin in Caenorhabditis elegans

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