Endogenous tryptophan residues of cAPK regulatory subunit type IIβ reveal local variations in environments and dynamics

Zawadzki, Kerri M.; Pan, Chia Pin; Barkley, Mary D.; Johnson, David A.; Taylor, Susan S.

doi:10.1002/prot.10326

Cited by 13 publications

(14 citation statements)

References 42 publications

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“…, or the intensity-weighted average fluorescence lifetime (28,60,61). These values represent the mean of the two or three reported sets of lifetimes Ϯ S.E.…”

Section: Table I Monoexponential and Double Exponential Fluorescence mentioning

confidence: 99%

Dynamics of Arrestin-Rhodopsin Interactions

Sommer

Smith

Farrens

2005

Journal of Biological Chemistry

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In this study, we address the mechanism of visual arrestin release from light-activated rhodopsin using fluorescently labeled arrestin mutants. We find that two mutants, I72C and S251C, when labeled with the small, solvent-sensitive fluorophore monobromobimane, exhibit spectral changes only upon binding light-activated, phosphorylated rhodopsin. Our analysis indicates that these changes are probably due to a burying of the probes at these sites in the rhodopsin-arrestin or phospholipid-arrestin interface. Using a fluorescence approach based on this observation, we demonstrate that arrestin and retinal release are linked and are described by similar activation energies. However, at physiological temperatures, we find that arrestin slows the rate of retinal release ϳ2-fold and abolishes the pH dependence of retinal release. Using fluorescence, EPR, and biochemical approaches, we also find intriguing evidence that arrestin binds to a post-Meta II photodecay product, possibly Meta III. We speculate that arrestin regulates levels of free retinal in the rod cell to help limit the formation of damaging oxidative retinal adducts. Such adducts may contribute to diseases like atrophic age-related macular degeneration (AMD). Thus, arrestin may serve to both attenuate rhodopsin signaling and protect the cell from excessive retinal levels under bright light conditions.The visual photoreceptor rhodopsin is perhaps the best model system for understanding the mechanisms used in Gprotein-coupled receptor (GPCR) 1 signaling, as detailed information exists on the structures and dynamic interactions of the protein constituents (1). Visual activation begins with absorption of light by the 11-cis-retinal chromophore in rhodopsin. The photoactivated form of rhodopsin, Rho* or "Meta II," interacts with and activates the G-protein transducin, which exchanges nucleotide and then diffuses to interact with downstream effectors. Signaling by Rho* is terminated by slow thermal decay and the release of retinal. Alternatively, signaling can be quickly terminated by a process that begins with phosphorylation of rhodopsin's C-terminal tail through the action of rhodopsin kinase (2, 3). The phosphorylated Rho* is then bound by arrestin, which stops signaling by physically occluding the G-protein binding site (4, 5).In the present study, we address how these two inactivation mechanisms are related and, specifically, what governs arrestin release from rhodopsin. Arrestin is known to bind to phosphorylated Meta II, a form in which the photolyzed chromophore all-trans-retinal is still attached to the receptor by a deprotonated Schiff base. Arrestin does not bind phosphorylated opsin, but all-trans retinal added exogenously can stimulate arrestin binding to phosphorylated opsin (6, 7). Early studies showed indirectly that retinal release and arrestin release are probably interrelated events (7,8). However, how these processes are linked or whether arrestin binds other photointermediates of rhodopsin (such as the storage form Meta III) is still unknown...

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“…, or the intensity-weighted average fluorescence lifetime (28,60,61). These values represent the mean of the two or three reported sets of lifetimes Ϯ S.E.…”

Section: Table I Monoexponential and Double Exponential Fluorescence mentioning

confidence: 99%

Dynamics of Arrestin-Rhodopsin Interactions

Sommer

Smith

Farrens

2005

Journal of Biological Chemistry

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“…The allosteric role is particularly apparent in the fluorescence spectra, which monitor the local environment of an A domain Trp reporter (32). Trp-243 is buried in a hydrophobic environment between the ␤ barrel and the helical subdomain of the A domain.…”

Section: Discussionmentioning

confidence: 99%

“…The mutations were confirmed by sequencing the entire plasmid. Expression and purification of wild-type RII␤ and a deletion mutant protein lacking the entire B domain was carried out as described previously (32). The two Arg mutant RII␤ proteins were purified by co-lysis with a poly-His-tagged C-subunit (33).…”

Section: Methodsmentioning

confidence: 99%

“…Urea Unfolding of the RII␤ Subunits-A stock solution of 8.5 M urea was prepared in buffer containing 25 mM sodium phosphate, 150 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, pH 7.4, as previously described (32). Proteins were unfolded in designated concentrations of urea for 2 h at room temperature and monitored by fluorescence.…”

Section: Methodsmentioning

confidence: 99%

“…Whereas RII␤ contains two endogenous tryptophans, Trp-58 in the linker region and Trp-243 in cAMP-binding domain A, the tryptophan fluorescence of wild-type RII␤ is due almost exclusively to Trp-243 (32). Fluorescence emission spectra of the wild-type, R230K, and R359K mutant proteins, thus, most likely reports the fluorescence emission from Trp-243 in the A domain.…”

Section: Local and Global Structural Changes Following Rii␤ Mutationsmentioning

confidence: 99%

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cAMP-dependent Protein Kinase Regulatory Subunit Type IIβ

Zawadzki

Taylor

2004

Journal of Biological Chemistry

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cAMP-dependent protein kinase (cAPK) contains a regulatory (R) subunit dimer bound to two catalytic (C) subunits. Each R monomer contains two cAMP-binding domains, designated A and B. The sequential binding of two cAMPs releases active C. We describe here the properties of RII␤ and two mutant RII␤ subunits, engineered by converting a conserved Arg to Lys in each cAMPbinding domain thereby yielding a protein that contains one intact, high affinity cAMP-binding site and one defective site. Structure and function were characterized by circular dichroism, steady-state fluorescence, surface plasmon resonance and holoenzyme activation assays. The K a for RII␤ is 610 nM, which is 10-fold greater than its K d (cAMP) and significantly higher than for RI␣ and RII␣. The Arg mutant proteins demonstrate that the conserved Arg is important for both cAMP binding and organization of each domain and that binding to domain A is required for activation. The K a of the A domain mutant protein is 21-fold greater than that of wild-type and the K d (cAMP) is increased 7-fold, confirming that cAMP must bind to the mutated site to initiate activation. The domain B mutant K a is 2-fold less than its K d (cAMP), demonstrating that, unlike RI␣, cAMP can access the A site even when the B site is empty. Removal of the B domain yields a K a identical to the K d (cAMP) of full-length RII␤, indicating that the B domain inhibits holoenzyme activation for RII␤. In RI␣, removal of the B domain generates a protein that is more difficult to activate than the wild-type protein.

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A multi‐angular mass spectrometric view at cyclic nucleotide dependent protein kinases: In vivo characterization and structure/function relationships

Scholten

Aye

Heck

2008

Mass Spectrometry Reviews

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Mass spectrometry has evolved in recent years to a well-accepted and increasingly important complementary technique in molecular and structural biology. Here we review the many contributions mass spectrometry based studies have made in recent years in our understanding of the important cyclic nucleotide activated protein kinase A (PKA) and protein kinase G (PKG). We both describe the characterization of kinase isozymes, substrate phosphorylation, binding partners and post-translational modifications by proteomics based methodologies as well as their structural and functional properties as revealed by native mass spectrometry, H/D exchange MS and ion mobility. Combining all these mass spectrometry based data with other biophysical and biochemical data has been of great help to unravel the intricate regulation of kinase function in the cell in all its magnificent complexity.

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Endogenous tryptophan residues of cAPK regulatory subunit type IIβ reveal local variations in environments and dynamics

Cited by 13 publications

References 42 publications

Dynamics of Arrestin-Rhodopsin Interactions

Dynamics of Arrestin-Rhodopsin Interactions

cAMP-dependent Protein Kinase Regulatory Subunit Type IIβ

A multi‐angular mass spectrometric view at cyclic nucleotide dependent protein kinases: In vivo characterization and structure/function relationships

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