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Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange

Geiser, A. H. and Sievert, M. K. and Guo, L. W. and Grant, J. E. and Krebs, M. P. and Fotiadis, D. and Engel, A. and Ruoho, A. E.. (2006) Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange. Protein Science, Vol. 15, H. 7. pp. 1679-1690.

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Official URL: http://edoc.unibas.ch/dok/A5262424

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Abstract

The mechanisms by which G-protein-coupled receptors (GPCRs) activate G-proteins are not well understood due to the lack of atomic structures of GPCRs in an active form or in GPCR/G-protein complexes. For study of GPCR/G-protein interactions, we have generated a series of chimeras by replacing the third cytoplasmic loop of a scaffold protein bacteriorhodopsin (bR) with various lengths of cytoplasmic loop 3 of bovine rhodopsin (Rh), and one such chimera containing loop 3 of the human beta(2)-adrenergic receptor. The chimeras expressed in the archaeon Halobacterium salinarum formed purple membrane lattices thus facilitating robust protein purification. Retinal was correctly incorporated into the chimeras, as determined by spectrophotometry. A 2D crystal (lattice) was evidenced by circular dichroism analysis, and proper organization of homotrimers formed by the bR/Rh loop 3 chimera Rh3C was clearly illustrated by atomic force microscopy. Most interestingly, Rh3C (and Rh3G to a lesser extent) was functional in activation of GTPgamma(35)S/GDP exchange of the transducin alpha subunit (Galphat) at a level 3.5-fold higher than the basal exchange. This activation was inhibited by GDP and by a high-affinity peptide analog of the Galphat C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the Galphat C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This Galphat-activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G-protein interactions.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Structural Biology (Engel)
UniBasel Contributors:Engel, Andreas H
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Cambridge University Press
ISSN:0961-8368
Note:Publication type according to Uni Basel Research Database: Journal article
Last Modified:22 Mar 2012 14:21
Deposited On:22 Mar 2012 13:24

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