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. 1999 Mar 2;96(5):2503-7.
doi: 10.1073/pnas.96.5.2503.

Gbeta5 prevents the RGS7-Galphao interaction through binding to a distinct Ggamma-like domain found in RGS7 and other RGS proteins

K Levay  1 , J L Cabrera, D K Satpaev, V Z Slepak
Affiliations

Gbeta5 prevents the RGS7-Galphao interaction through binding to a distinct Ggamma-like domain found in RGS7 and other RGS proteins

K Levay et al. Proc Natl Acad Sci U S A. .

Abstract

The G protein beta subunit Gbeta5 deviates significantly from the other four members of Gbeta-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gbeta5 in vivo, we have isolated a native Gbeta5 protein complex from the retinal cytosolic fraction and identified the protein tightly associated with Gbeta5 as the regulator of G protein signaling (RGS) protein, RGS7. Here we show that complexes of Gbeta5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gbeta5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gbeta5 or anti-RGS7 antibodies. The specific Gbeta5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Ggamma subunits. Deletion of this domain prevents the RGS7-Gbeta5 binding, although the interaction with Galpha is retained. Substitution of the Ggamma-like domain of RGS7 with a portion of Ggamma1 changes its binding specificity from Gbeta5 to Gbeta1. The interaction of Gbeta5 with RGS7 blocked the binding of RGS7 to the Galpha subunit Galphao, indicating that Gbeta5 is a specific RGS inhibitor.

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Figures

Figure 1
Figure 1
Gβ5-RGS7 interaction in vitro. (Upper) Overlay plot of three experiments resolving monomeric Gβ5 (■), Gβ5 with excess Gγ2 (□, gray line), and monomeric RGS7 (▴) on a Superdex 200 gel-filtration column as described in Materials and Methods. The G protein γ subunit Gγ2 was synthesized in the presence of nonradioactive methionine. The Gβ5γ2 complex has a lower apparent molecular weight than Gβ5 apparently because of a more compact structure (20). (Lower) Experiment with the mixture of Gβ5 with RGS7 (squares, position of Gβ5; triangles, RGS7). x axis: Elution volume (ml), starting (zero) at the beginning of elution of the blue dextran. Highlighted area below the axis denotes the fractions resolved by SDS/PAGE and radioautography, shown to the right. y axis: Arbitrary units based on the strength of the bands on the gel determined by the amount of pixels per band. The fractions were analyzed by SDS/PAGE followed by radioautography, and the amount of 35S-labeled Gβ5 or RGS7 was measured by image analysis of the exposed film using the nih image software. Each experiment was done at least two times, each with an independent in vitro translation.
Figure 2
Figure 2
Analysis of RGS7-Gβ complex formation in vitro by cation-exchange chromatography and immunoprecipitation. (A) Chromatography on Sepharose S. The lysates containing [35S]Met-labeled Gβ5, Gβ1, RGS7, or their mixture were incubated batchwise with the chromatography resin. The unbound material was collected, the beads then were washed and eluted by 300 mM of NaCl, and proteins from the fractions were analyzed by SDS/PAGE. T, total lysate loaded; U, unbound material; W, washes; E, the eluate. (B) The Gβ5γ2 complex was obtained by mixing the 35S-labeled Gβ5 and the excess of unlabeled Gγ2 under the same conditions as in Fig. 1. 35S-RGS7-containing lysate then was added to the mixture, and binding to Sepharose S was tested as in A. (C) Immunoprecipitation. The antibodies indicated were adsorbed on protein A-Sepharose, and the lysates were added to the beads. After incubation, the beads were washed and eluted by SDS, and the obtained fractions were processed as in A. T, total mixture added; W, washes; E, eluate.
Figure 3
Figure 3
Structural homology between Gγ subunits and RGS proteins. Alignment of full-length sequences of Gγ subunits and the indicated portions of RGS proteins. Asterisks above the Gγ sequences designate the residues that are identical throughout the entire Gγ class and the residues found at corresponding positions in at least one RGS protein. Boxed are the regions of homology based on the nature of amino acids, i.e., basic (K, R) acidic (E, D), hydrophobic (L, I, V, F, W, M), polar (S, T), and amides (Q, N). EGL-10 has an 8-residue insert shown below its sequence. The sequence highlighted in RGS7 was deleted in the RGS7Δ mutant (see text and Fig. 4) or swapped for the stretch of Gγ1 amino acids highlighted in Gγ1.
Figure 4
Figure 4
Functional activity of Gγ-like domain in RGS7. (A) Structural domains in wild-type RGS7 and its mutants. The thinner bar represents the length of the protein. The stippled box is the RGS core domain; the solid box denotes the Gγ-like domain. The open box is the “DEP,” which also is found in RGS6, 7, and 9 and EGL-10; its sequence is homologous to pleckstrin (29) but the function is unknown. In the RGS7Δ mutant, the Gγ-like domain is deleted, and in the RGS7γ1 mutant, it is replaced with a portion of Gγ1 (hatched box). (B) Interaction of the mutants with Gβ5 and Gβ1. The experiments were carried out by using the 35S-labeled proteins and testing the Gβ5-RGS interaction using Sepharose S as described in the Fig. 2 legend. Gβ5 binds the wild-type RGS7 (Top), but not the RGS7Δ mutant (Middle). (Bottom) RGS7γ1 mutant binds Gβ1 instead of Gβ5.
Figure 5
Figure 5
Gβ5 inhibits the interaction between RGS7 and Gαo. His-tagged Gαo (30) was immobilized on the Ni2+-NTA beads, and the 35S-labeled RGS7 or its mixture with Gβ5 was applied, batchwise, to the suspension in the presence of 0.1 mM GDP/0.1 mM GDP plus 10 mM NaF and 100 μM AlCl3 or guanosine 5′-[γ-thio]triphosphate. The beads were washed and eluted with sample buffer for SDS/PAGE. The resin without Gαo was used for control of a nonspecific adsorption. (A) Radioautograms of the fractions from the chromatography resolved by SDS/PAGE followed by radioautography. (B) The amount of the protein in the bands was quantified by the image analysis of the exposed film. The bar graph shows the amount of RGS7 eluted from immobilized (His)6-Gαo or control Ni2+ beads without Gαo. Data were collected from three independent experiments.
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