Journal
PROTEIN SCIENCE
Volume 15, Issue 7, Pages 1679-1690Publisher
WILEY
DOI: 10.1110/ps.062192306
Keywords
G-protein-coupled receptors; chimera; bacteriorhodopsin; rhodopsin; transducin; GTP exchange
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Funding
- NCRR NIH HHS [S10 RR13790, S10 RR013790] Funding Source: Medline
- NIGMS NIH HHS [GM33138, R01 GM033138] Funding Source: Medline
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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 GTP gamma S-35/GDP exchange of the transducin a subunit ( Gat) 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 Gat C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the Gat C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This G alpha t-activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G-protein interactions.
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