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

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 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.

Figure 1.

Schematic of bR/Rh chimera construction. Shown is the bR/Rh chimera, Rh3C. Amino acid residue numbers are noted for bR (Luecke et al. 1999) and Rh (Palczewski et al. 2000). Shaded amino acids are the Rh residues added into the bR sequence. The Rh residues were added onto the ends of the corresponding bR helices (shown as dotted lines in the helices). The Rh residues shown in brackets are the amino acids that were systematically removed from each end of the insert, which resulted in the other bR/Rh chimeric constructs with shorter inserts (Table 1) and thus different “twisting” of the helical region of rhodopsin. To distinguish the nomenclatures of the bR and Rh transmembrane helices, we use A–G for bR helices and 1–7 for Rh helices.

Figure 2.

Purification of bR/GPCR chimeras expressed in H. salinarum PM. (A) Purple phenotype of a bR/Rh chimera (only Rh3C data are presented here). A bR-deficient strain of H. salinarum, MPK40, was transformed with the chimeric gene through a homologous recombination protocol (Peck et al. 2000). The transformed cells were plated on a mevinolin selection media, and the plate was incubated at 37°C under illumination for 3 d. A purple colony, indicating proper formation of the bR lattice structure, and a wild-type colony with no transformation (not expressing any bR), are indicated by arrows. (B) Wild-type bR, bR/GPCR chimeras Rh3C and β₂L3 were collected from H. salinarum cells and purified by equilibrium centrifugation over a sucrose density gradient. The PM migrated in the middle of the tube as a purple band. (C) Purified bR and chimeras were incubated with Laemmli buffer containing SDS and DTT at room temperature overnight, and then subjected to 12% SDS-PAGE. Rh3G ran at the same position as Rh3C (data not shown).

Figure 3.

Spectroscopic characterization of bR/GPCR chimeras. The spectroscopic properties of the purified bR and bR/GPCR chimeras were compared, by scanning their UV/Vis absorbance (A) and CD spectroscopy (B). The experiments were performed as described in Materials and Methods. As can be seen in this figure, bR and the chimeras exhibit similar spectra.

Figure 4.

AFM imaging of Rh3C purple membrane. The AFM topograph of Rh3C extracellular surface shows that the homotrimers of chimera Rh3C are well organized in the 2D crystal. A trimer of Rh3C is magnified as shown in the circle. AFM imaging was conducted as described in Materials and Methods.

Figure 5.

GTPγ³⁵S exchange assay showing activation of transducin by the bR/Rh chimeras. (A) The activation of transducin by the purified bR or chimeras Rh3C and Rh3G was assayed by incubating the chimeras (0.6 μM each) with 0.2 μM transducin and 5 μM GTPγ³⁵S for 10 min at 30°C. The samples were rapidly vacuum-filtered through Millipore cellulose acetate filters (0.22-μm pore size). The ³⁵S content on the filters was determined by liquid scintillation counting. The basal activation is represented by the condition of holoT alone (one fold; first bar). Native rhodopsin was used for the assay as a positive control (last bar), at a molar ratio of 2.3:1 vs. transducin. All the assays were performed under room light. Error bars represent standard deviations (SDs). (B) Chimera Rh3C-catalyzed GTPγ³⁵S exchange on transducin was completely inhibited by the addition of GDP in a dose-dependent manner, but to a much less extent by ADP. Data are presented as the percentage of the GTPγ³⁵S exchange control without adding any nucleotide. (C) Chimera Rh3C-catalyzed GTPγ³⁵S exchange on transducin was inhibited by the addition of a high affinity transducin C-terminal peptide analog (termed VLED) identified by Martin et al. (1996), but only minimally by a randomized version of the peptide. Data are presented as the percentage of the GTPγ³⁵S exchange in the absence of any peptide (the top line).

Figure 6.

Transducin activation by bR/Rh chimeras assayed by photolabeling using [³²P]-(4-azidoanilido)-GTP. Holotransducin was incubated with the photoactivatable GTP analog, [³²P]-(4-azidoanilido)-GTP, either alone or in the presence of purified chimeras (Rh3C or Rh3G) at an approximate molar ratio of 1:1, as can be seen from the Coomassie-stained bands (left panel). The samples were photoactivated using a 1-kW mercury vapor lamp from a distance of 10 cm. Samples were electrophoresed on a 12% SDS-polyacrylamide gel, and radioactivity was measured using a Molecular Dynamics PhosphorImager (pseudocolor is depicted; right panel). The photolabeling of Gαt was quantitated using ImageQuant and plotted as fold labeling with each bar (±SD) on top of its corresponding triplet lanes.

Figure 7.

Cocentrifugation of transducin and chimera Rh3C. (A) Cocentrifugation of transducin with bR (lane 1) and Rh3C (lane 3) was detected by Western blotting. The Gαt C terminus analog VLED (in 10-fold molar excess over Gαt) protected pull-down of transducin by Rh3C (lane 4) but not the background by bR (lane 2). (B) Quantitation of transducin pull-down. The intensity of Western signal from Gαt is normalized by the amount of protein in the bR or Rh3C band as detected by Coomassie staining. Each bar represents an average (±SD) of three lanes, and all the lanes are from the same blot. (*) p < 0.05. (C) Cocentrifugation of transducin with V8 protease-cleaved Rh3C (lane 3) is compared to that with uncleaved Rh3C (lane 2) and bR (lane 1). Western blotting was performed as in A. (D) A Coomassie-stained SDS gel (16%) shows cleavage of Rh3C loop 3 by V8 protease. Lane 3 indicates that Rh3C was cleaved into two bands, a higher band (∼18 kDa) and a lower one (∼6 kDa). bR (lane 1) and Rh3C (lane 2) were also run on the same gel to indicate the position of uncleaved Rh3C.