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Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange
Author(s) -
Geiser Andrew H.,
Sievert Michael K.,
Guo LianWang,
Grant Jennifer E.,
Krebs Mark P.,
Fotiadis Dimitrios,
Engel Andreas,
Ruoho Arnold E.
Publication year - 2006
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.062192306
Subject(s) - transducin , g protein coupled receptor , rhodopsin , g protein , bacteriorhodopsin , chimera (genetics) , halobacterium salinarum , gtp' , chemistry , mastoparan , cytoplasm , biology , biophysics , biochemistry , receptor , membrane , retinal , gene , enzyme
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 β 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 GTPγ 35 S/GDP exchange of the transducin α subunit (Gαt) 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 Gαt C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the Gαt C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This Gαt‐activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G‐protein interactions.