Site of G Protein Binding to Rhodopsin Mapped with Synthetic Peptides from the α Subunit

Hamm, Heidi E.; Deretic, Dusanka; Arendt, Anatol; Hargrave, Paul A.; Koenig, Bernd W.; Hofmann, Klaus Peter

doi:10.1126/science.3136547

Cited by 504 publications

(396 citation statements)

References 26 publications

Supporting

Mentioning

374

Contrasting

Unclassified

Order By: Relevance

“…As key Meta II binding site on the G protein, the C-terminus of the G t α subunit was found [153,154,156,157]. Biochemical, biophysical and mutagenesis studies [158] provided evidence that the G t α C terminus might bind into the cytoplasmic crevice of the 'blossomed' opsin* (Figure 3.9).…”

Section: ) Conformational Similarity Between Opsin* and Opsin*•g T αmentioning

confidence: 99%

See 1 more Smart Citation

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Park

Scheerer

Hofmann

et al. 2008

Nature

842

925

View full text Add to dashboard Cite

Compared with the known structure of inactive rhodopsin, opsin displays prominent structural changes in the conserved E(D)RY and NPxxY(x) 5,6 F regions and TM5-TM7. At the cytoplasmic side, TM6 is tilted outwards by 6-7 Å, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, of which some are attributed to an active GPCR state, reorganize the empty retinal binding pocket to disclose two openings for exit and entry of retinal. The opsin structure thus sheds new light on binding of hydrophobic ligands to GPCRs, GPCR activation and signal transfer to the G protein.

show abstract

Section: ) Conformational Similarity Between Opsin* and Opsin*•g T αmentioning

confidence: 99%

“…Previous protein-protein interaction studies identified Meta II as interaction partner for transducin and fragments thereof [72,86,123,134,152,153,154,155]. As key Meta II binding site on the G protein, the C-terminus of the G t α subunit was found [153,154,156,157].…”

Section: ) Conformational Similarity Between Opsin* and Opsin*•g T αmentioning

confidence: 99%

Crystal structure of the ligand-free G-protein-coupled receptor opsin

Park

Scheerer

Hofmann

et al. 2008

Nature

842

925

View full text Add to dashboard Cite

show abstract

“…Alternative approaches have provided some insights into the sites in rhodopsin required for binding of T. Thus, peptides corresponding to the amino acid sequences in the different cytoplasmic loops of rhodopsin inhibit T activation (2), and similarly mutagenic studies (3)(4)(5) have indicated that a considerable portion of the cytoplasmic domain of light-activated rhodopsin is involved in binding to T. Some information also has been obtained on the regions of T that contact rhodopsin in the complex. Thus, the effects of anti-T ␣ antibody binding (6,7), ADP-ribosylation of the ␣ subunit of transducin (T ␣ ), and peptide competition for binding (8) all indicated that the C-terminal region of T ␣ is a major site of interaction with rhodopsin. More recently, involvement of amino acid sequence 330-350 in the C-terminal region of T ␣ in binding to rhodopsin received further support by alanine scanning, chimeric receptor studies, and peptide-binding studies (9)(10)(11).…”

mentioning

confidence: 99%

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Cai

Itoh²,

Hg³

2001

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

140

View full text Add to dashboard Cite

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfideexchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at >495 nm. Subsequent irradiation of the complex at 310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with T ␣. For identification of the sites of crosslinks in T ␣, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the T ␣ species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the Nmaleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting T ␣ derivatives and isolation of T␣ peptides carrying maleimido-butyrylbiocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption͞ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in T ␣ containing the amino acid sequences 310 -313 and 342-345.L ight activation of rhodopsin initiates two biochemical cascades, one leading to sensitization (amplification) and the other to desensitization (quenching). Binding and activation of transducin (T) and rhodopsin kinase are, respectively, the first events in the two cascades (1). Competition between T and rhodopsin kinase for interaction with the light-activated rhodopsin and its phosphorylated form is central to the progression of the two cascades. Therefore, understanding the requirements of the two proteins for binding to rhodopsin and the contact sites in the complexes formed is of much interest. Ideally, three-dimensional structures of the complexes between the proteins are needed, but such structural analysis lies in the future. Alternative approaches have provided some insights into the sites in rhodopsin required for binding of T. Thus, peptides corresponding to the amino acid sequences in the different cytoplasmic loops of rhodopsin inhibit T activation (2), and similarly mutagenic studies (3-5) have indicated that a considerable portion of the cytoplasmic domain of light-activated rhodopsin is involved in binding to T. Some information also has been obtained on the regions of T that contact rhodopsin in the complex. Thus, the effects of a...

show abstract

“…Thus, we cannot expect physiological function of HrGα n by analogy. On this issue it should be noted that the amino acid sequence of HrGα n was quite unique in its C-terminal region, which is a possible interaction site of G protein α subunit with its receptor molecule (Hamm et al, 1988;Nishimura et al, 1998;Gilchrist et al, 2002). It can be expected that HrGα n couple with a novel receptor.…”

Section: Discussionmentioning

confidence: 99%