G-protein-effector coupling: A real-time light-scattering assay for transducin-phosphodiesterase interaction

Heck, M.; Hofmann, Klaus Peter

doi:10.1021/bi00083a024

Cited by 100 publications

(53 citation statements)

References 50 publications

(92 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The materials and proteins used were as described (17,18). Purified antirhodopsin mAb rho-1D4 was obtained from the National Culture Center, Minneapolis.…”

Section: Methodsmentioning

confidence: 99%

Role of the conserved NPxxY(x) _5,6 F motif in the rhodopsin ground state and during activation

Fritze

Filipek

Kuksa

et al. 2003

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

Self Cite

334

315

View full text Add to dashboard Cite

In the G protein-coupled receptor rhodopsin, the conserved NPxxY(x) 5,6F motif connects the transmembrane helix VII and the cytoplasmic helix 8. The less geometrically constrained retinal analogue 9-demethyl-retinal prevents efficient transformation of rhodopsin to signaling metarhodopsin (Meta) II after retinal photoisomerization. Here, we demonstrate that Ala replacement mutations within the NPxxY(x) 5,6F domain, which eliminate an interaction between aromatic residues Y306 and F313, allow formation of Meta II despite the presence of 9-demethyl-retinal. Also a disulfide bond linking residues 306 and 313 in the 9-demethylretinal-reconstituted mutant Y306C͞F313C͞C316S prevented Meta II formation, whereas the reduced form of the mutant readily transformed to Meta II after illumination. These observations suggest that the interaction between residues 306 and 313 is disrupted during the Meta I͞Meta II transition. However, this enhancement in Meta II formation is not reflected in the G protein activation, which is dramatically reduced for these mutants, suggesting that changes in the Y306 -F313 interaction also lead to a proper realigning of helix 8 after photoisomerization. The E134Q mutation, located in the second conserved motif, D(E)RY, rescues activity in 9-demethyl-retinal-reconstituted mutants to different degrees, depending on the position of the Ala replacement in the NPxxY(x) 5,6F motif, thus revealing distinct roles for the NP and Y(x) 5,6F portions. Our studies underscore the importance of the NPxxY(x) 5,6F and D(E)RY motifs in providing structural constraints in rhodopsin that rearrange in response to photoisomerization during formation of the G protein-activating Meta II. The dual control of the structural rearrangements secures reliable transformation of quiescent rhodopsin to activating Meta II.GPCR ͉ NPxxY motif R hodopsin is a prototypical receptor from the largest subfamily A of G protein-coupled receptors (GPCRs), which are thought to operate through similar signaling mechanisms (1, 2). The only known crystal structure of a GPCR, that of rhodopsin (3), confirmed the presence of seven transmembrane helices (H). The structure describes the receptor in its inactive ground state with the bound inverse agonist, 11-cis-retinal. The -amino group of K296 in H-VII tethers the chromophore 11-cis-retinal to opsin via a covalent Schiff base (SB) bond (Fig. 1). A salt-bridge between the protonated SB and the counterion, the conserved Glu-113 in H-III, contributes largely to constrain the receptor in the inactive conformation. Additional constraints involve hydrogen bond networks and hydrophobic interactions linking transmembrane helices, sequestration of the Arg residue of the D(E)RY motif in the hydrophobic milieu, and intramolecular interactions within the NPxxY(x) 5,6 F region (3, 4). Absorption of light energy isomerizes the 11-cis-retinylidene chromophore, generating the agonist all-trans-retinylidene in situ. Subsequent thermal relaxation of the retinal-protein complex leads within milliseconds to an e...

show abstract

“…The materials and proteins used were as described (17,18). Purified antirhodopsin mAb rho-1D4 was obtained from the National Culture Center, Minneapolis.…”

Section: Methodsmentioning

confidence: 99%

Role of the conserved NPxxY(x) _5,6 F motif in the rhodopsin ground state and during activation

Fritze

Filipek

Kuksa

et al. 2003

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

Self Cite

334

315

View full text Add to dashboard Cite

show abstract

“…To remove retinaloxime from the membrane-bound phosphorylated opsin, the membranes were treated with urea and fatty acid-free bovine serum albumin (26). An average stoichiometry of ϳ1.5 phosphates/opsin was determined using radioactive [␥- 32 P]ATP as a tracer. Phosphorylated rhodopsin was prepared by regeneration of phosphorylated opsin with 11-cis-retinal (27).…”

Section: Methodsmentioning

confidence: 99%

“…Transducin was purified from frozen dark-adapted bovine retinas (32). Purified transducin (G t ) concentration was determined using the Bradford method (33).…”

Section: Methodsmentioning

confidence: 99%

Arrestin and Its Splice Variant Arr1–370A(p44)

Schröder

Pulvermüller

Hofmann

2002

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

“…Preparation of Transducin (G t )-G t was purified from bovine retinae essentially as described (30) and stored in 20 mM BTP, pH 7.1, 130 mM NaCl, 1 mM MgCl 2 , 1 mM dithiothreitol.…”

Section: Methodsmentioning

confidence: 99%

Signaling States of Rhodopsin

Meyer¹,

Böhme²,

Ockenfels³

et al. 2000

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After lightinduced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.The retinal photoreceptor rhodopsin is one of the archetypes of the G-protein-coupled receptor superfamily. It is composed of the apoprotein opsin comprising seven transmembrane helices (TMs) 1 and the chromophore 11-cis-retinal, which is covalently bound to Lys 296 in TM7 via a protonated Schiff base (SB), keeps the receptor in the inactive conformation, and acts as a highly effective inverse agonist. Following the absorption of a photon, the retinal isomerizes to a strained all-trans-conformation (1), which induces a series of conformational rearrangements of the opsin moiety. The final product of this reaction sequence is the active conformation (R*) that contains all-trans-retinal as a covalently bound agonist and is capable of catalyzing the activation of the retinal G-protein transducin (G t ) (for reviews see Refs. 2-4).These events can be summarized as shown in Fig. 1A). After cis-trans-isomerization and initial, short lived intermediates, illuminated rhodopsin progresses from the lumirhodopsin form to the first long lived state, metarhodopsin I (meta I). Up to and including meta I, the SB of the pigment remains protonated. Deprotonation of the SB and protonation of its counterion Glu 113 mark the transition from the meta I ( max ϭ 478 nm) to the metarhodopsin II (meta II) conformation (5), which is characterized by a strongly blue-shifted absorbance maximum ( max ϭ 380 nm). SB deprotonation is known to be necessary for interaction with G t (6 -8) and is accompanied by the uptake of a proton from the aqueous solution (9 -11). pH rate profiles of G t activation and proton uptake measurements have shown the opsin residue Glu 134 to be an important mediator of the uptake or even the acceptor of this proton (12, 13). Based on computer ...

show abstract

G-protein-effector coupling: A real-time light-scattering assay for transducin-phosphodiesterase interaction

Cited by 100 publications

References 50 publications

Role of the conserved NPxxY(x) _5,6 F motif in the rhodopsin ground state and during activation

Role of the conserved NPxxY(x) _5,6 F motif in the rhodopsin ground state and during activation

Arrestin and Its Splice Variant Arr1–370A(p44)

Signaling States of Rhodopsin

Contact Info

Product

Resources

About

G-protein-effector coupling: A real-time light-scattering assay for transducin-phosphodiesterase interaction

Cited by 100 publications

References 50 publications

Role of the conserved NPxxY(x) 5,6 F motif in the rhodopsin ground state and during activation

Role of the conserved NPxxY(x) 5,6 F motif in the rhodopsin ground state and during activation

Arrestin and Its Splice Variant Arr1–370A(p44)

Signaling States of Rhodopsin

Contact Info

Product

Resources

About

Role of the conserved NPxxY(x) _5,6 F motif in the rhodopsin ground state and during activation

Role of the conserved NPxxY(x) _5,6 F motif in the rhodopsin ground state and during activation