Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange.
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 beta2-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 GTPgamma35S/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.
Geiser
Andrew H
AH
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706, USA.
Sievert
Michael K
MK
Guo
Lian-Wang
LW
Grant
Jennifer E
JE
Krebs
Mark P
MP
Fotiadis
Dimitrios
D
Engel
Andreas
A
Ruoho
Arnold E
AE
D000818:Animals; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D002417:Cattle; D003593:Cytoplasm; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012172:Retinaldehyde / Q000737:chemistry; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics; D015293:Transducin / Q000378:metabolism*
D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D001436:Bacteriorhodopsins; D006160:Guanosine Triphosphate; D012243:Rhodopsin; D015293:Transducin; D012172:Retinaldehyde