Rhodopsin activation exposes a key hydrophobic binding site for the transducin α-subunit C terminus

Jay M. Janz, David Farrens

Research output: Contribution to journalArticle

90 Citations (Scopus)

Abstract

Conformational changes enable the photoreceptor rhodopsin to couple with and activate the G-protein transducin. Here we demonstrate a key interaction between these proteins occurs between the C terminus of the transducin α-subunit (G) and a hydrophobic cleft in the rhodopsin cytoplasmic face exposed during receptor activation. We mapped this interaction by labeling rhodopsin mutants with the fluorescent probe bimane and then assessed how binding of a peptide analogue of the G. C terminus (containing a tryptophan quenching group) affected their fluorescence. From these and other assays, we conclude that the G C-terminal tail binds to the inner face of helix 6 in a retinal-linked manner. Further, we find that a "hydrophobic patch" comprising key residues in the exposed cleft is required for transducin binding/activation because it enhances the binding affinity for the G C-terminal tail, contributing up to 3 kcal/mol for this interaction. We speculate the hydrophobic interactions identified here may be important in other GPCR signaling systems, and our Trp/bimane fluorescence methodology may be generally useful for mapping sites of protein-protein interaction.

Original languageEnglish (US)
Pages (from-to)29767-29773
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number28
DOIs
StatePublished - Jul 9 2004

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Transducin
Rhodopsin
Chemical activation
Binding Sites
Fluorescence
Proteins
Hydrophobic and Hydrophilic Interactions
Fluorescent Dyes
GTP-Binding Proteins
Tryptophan
Labeling
Quenching
Assays
Peptides
bimanes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Rhodopsin activation exposes a key hydrophobic binding site for the transducin α-subunit C terminus. / Janz, Jay M.; Farrens, David.

In: Journal of Biological Chemistry, Vol. 279, No. 28, 09.07.2004, p. 29767-29773.

Research output: Contribution to journalArticle

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