Perturbing the Linker Regions of the α-Subunit of Transducin

Abstract

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the ␣ subunits of transducin (␣ T ) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a "clam shell" that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve "opening the clam shell" to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of G␣ subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an ␣ T /␣ i1 chimera (designated ␣ T * ). Both ␣* T linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal ␣ T or recombinant ␣ * T . The ␣ * T linker mutants were responsive to aluminum fluoride, which binds to ␣-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the ␤␥ complex, their activation was not influenced by ␤␥ alone, arguing against the idea that the ␤␥ complex helps to pry apart the helical and Ras-like domains of G␣ subunits. Once activated, the ␣* T linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated G␣ mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.The visual phototransduction cascade in retinal rod outer segments consists of the heterotrimeric G protein, transducin, its upstream receptor rhodopsin and its downstream effector, cGMP-phosphodiesterase (PDE).1 This is a highly sensitive system, capable of detecting and being activated by a single photon of light, which excites the seven-membrane spanning receptor, rhodopsin. Upon the absorption of light, rhodopsin is activated through the photoisomerization of the chromophore, retinal, which is attached through a Schiff base linkage to the seventh membrane-spanning helix of the protein. Light-activated rhodopsin binds and activates the ␣ subunit of transducin (␣ T ) by stimulating its GDP-GTP exchange reaction. Activated GTP-bound ␣ T then stimulates PDE activity by altering the positions of its regulatory ␥ subunits (␥ PDE ) and thereby allowing the ␣ and ␤ catalytic subunits (␣ PDE and ␤ PDE ) to hydrolyze and consequently decrease cellular cGMP concentrations. This causes cation-specific cGMP-gated ion channels in the outer segments to close, leading to the hyperpolarization of rod outer segment membranes and the generation of the visual response. There is an amplification of the signal at every step of this cascade such that a single photon of light can lead to the inhibition of 10 6 -10 7 Na ϩ ions from entering the rod cell. Members of the family of large G proteins are made up of three ...

Mapping allosteric connections from the receptor to the nucleotide-binding pocket of heterotrimeric G proteins

“…Nearly every site examined in the switch I region demonstrated receptor activationdependent conformational changes. In addition, there are increases in the basal nucleotide exchange for sites 171R1, 182R1, and 184R1, consistent with previous studies by Majumdar et al (24) showing that Gly3Pro mutations in switch I increase basal GDP release rates in a G␣t/G␣i chimera. Comparing the heterotrimeric versus receptor-bound EPR spectra for sites 182R1, 184R1, and 186R1 near the G␤ interface demonstrates a clear structural rearrangement of the G␣i-G␤ interface.…”

“…The GTPase and helical domains clamp down on the guanine nucleotide, and it has been speculated that to exchange guanine nucleotides there must be a conformational change that opens the cleft (3,24,25). Switch I is one of the linkers between the two domains and is connected to the receptor-binding domain at the C terminus and the ␤2-␤3 loop by the ␤2-strand.…”

A Dominant-negative Gα Mutant That Traps a Stable Rhodopsin-Gα-GTP-βγ Complex

“…3B, inset, open triangles). This rate enhancement due to light-activated rhodopsin is similar to what has been reported earlier (10). However, the addition of even relatively high levels of R* to the ␣ T *(S43N) mutant had only minimal effects on the rate of nucleotide exchange (Fig.…”

“…4A, inset). This rate enhancement, due to the ␤1␥1 complex, is similar to that reported in earlier studies (10). However, this was not the case when assaying the ␣ T *(S43N) mutant, as the rate of nucleotide exchange upon the addition of 400 nM ␤1␥1 to ␣ T *(S43N) in the presence of 14 nM R* was 0.11 min Ϫ1 (Fig.…”

New Insights into the Role of Conserved, Essential Residues in the GTP Binding/GTP Hydrolytic Cycle of Large G Proteins

“…Protein Expression and Purification-The recombinant wild-type ␣ T * subunit and the ␣ T * mutants were expressed in BL21 (DE3) supercompetent cells and purified as described (18,19). The proteins were further purified by gel filtration chromatography on a HiLoad Superdex 75 HR26/60 column equilibrated with a buffer containing 20 mM NaHepes, pH 7.5, 5 mM MgCl 2 , and 1 mM NaN 3 .…”