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S. A. Vishnivetskiy, D. J. Francis, S. M. Hanson, N. Van Eps, W. L. Hubbell, C. S. Klug, V. V. Gurevich; Arrestin Alpha-Helix I Participates in Rhodopsin Binding: What Does the Complex Look Like?. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1670.
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Identification of receptor-binding elements of visual arrestin and elucidation of the shape of the arrestin-rhodopsin complex.
Site-directed mutagenesis, site-directed spin labeling EPR spectroscopy, and long range intramolecular distance measurements with DEER were used.
We found that alpha-helix I, localized in the arrestin N-domain, participates in rhodopsin binding. Mutations in the helix significantly reduce arrestin binding to phosphorylated light-activated rhodopsin and decrease the stability of the arrestin-rhodopsin complex. The analysis of accessibility of spin labels introduced in the helix for hydrophilic and hydrophobic reagents shows that the helix is in the hydrophilic environment in free and rhodopsin-bound arrestin, excluding its interaction with the membrane as proposed earlier. Distance measurements by EPR in doubly spin labeled arrestin show that in the rhodopsin-bound and free arrestin the position of the helix remains essentially the same.
Based on the localization of previously identified rhodopsin-binding elements to the concave surfaces of the two arrestin domains, it was generally believed that in the arrestin-receptor complex these concave sides of arrestin face the plane of the membrane. Direct participation of alpha-helix I in rhodopsin binding would be incompatible with this model. Our recent finding that each receptor molecule binds its own arrestin excludes the possibility that the concave sides of the two domains and the helix interact with two different receptor molecules. We propose two models of the arrestin-rhodopsin complex consistent with direct and indirect roles of alpha-helix I in the interaction. Direct distance measurements between selected positions in arrestin and rhodopsin are necessary to distinguish between these two models.
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