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M. Downs, R. Arimoto, G.R. Marshall, O. Kisselev; Cysteine Modifications on Rhodospin Result in Selective Interactions with the C-terminal Domains of Transducin Alpha- and Gamma-subunits . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1512.
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© ARVO (1962-2015); The Authors (2016-present)
Photoactivated rhodopsin interacts with domains on all three subunits of transducin. Two of these domains, C-terminal regions of the alpha- and gamma-subunit stabilize metarhodopsin II, but display different roles in the transducin activation process. Whether the interactions are with the same or different sites on Meta II is unknown. We have used difference UV/Visible spectroscopy to examine whether chemical modifications of the Cysteine 140 and Cysteine 316 with a combination of N-ethyl-maleimide and iodoacetamidosalicilate lead to selective disruption of interactions between Meta II and model synthetic peptides from transducin alpha- and gamma-subunits. Under conditions that favor spontaneous Meta II, modifications of Cysteine140 or Cysteine 316 did not impede the ability of photoactivated rhodopsin to form this photointermediate. However, G-protein domains Gt-alpha-(340-350) and Gt-gamma-(50-71)farnesyl showed drastically different patterns of interactions with Meta II. Neither double, nor single labeling of Cysteine140 and Cysteine 316 affected the interactions between Meta II and the gamma-peptide. In contrast, thioalkylation of Cysteine 140 by NEM abolished interactions between Meta II and the alpha-peptide almost completely. Cysteine 316-modified rhodopsin retained the ability to bind the alpha-peptide. These results suggest that formal structural determinants on Meta II for binding Gt-alpha-(340-350) and Gt-gamma-(50-71)farnesyl of transducin are different. Cysteine 140 appears to be a part of the binding site for the alpha-subunit. Neither one of the Cysteine140 or Cysteine 316 contribute to the binding site for the gamma-subunit. This work is supported by NIH GM63203 and American Heart Association (to O.G.K.), and NIH EY012113 (to G.R.M.).
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