1. Double Suppression of the Gα Protein Activity by RGS Proteins
Chen Lin, Alexey Koval, Svetlana Tishchenko, Azat Gabdulkhakov, Uliana Tin, Gonzalo P. Solis, Vladimir L. Katanaev 
Molecular Cell 
Volume 53, Issue 4, Pages (February 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 53, 663-671DOI: (10.1016/j.molcel.2014.01.014)
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3. Figure 1
Dhit Has Two Gαo-Binding Regions and Interacts with Different Nucleotide Forms of Gαo
(A) Dhit contains a conserved cysteine-rich string and a catalytic RGS domain (amino acids 142–257). In the Y2H assay, Gαo[Q205L]-interacting region of Dhit spans amino acids 126–251, while fragments of Dhit interacting with the wild-type Gαo include amino acids 122–256.
(B) In pull-down experiments, Dhit is efficiently coprecipitated by matrix-immobilized Gαo preloaded either with GDP, GTPγS, or GDP-AlF4−. Control matrix with immobilized MBP-βgal does not bind Dhit.
(C) The C-terminal (RGS-containing, aa 105–274) and N-terminal (aa 1–129) parts of Dhit both interact with Gαo. While C-Dhit preferentially interacts with Gαo-GTPγS, N-Dhit indiscriminately binds both nucleotide forms of the G protein.
(D) Binding of C-Dhit to Gαo-GDP is not outcompeted by increasing concentrations of N-Dhit. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Dhit Exerts GAP and GDI Activity toward Gαo
(A and B) BODIPY-GTP was used to probe the GAP and GDI activities of Dhit toward Gαo, showing an example of individual BODIPY-GTP uptake and hydrolysis curves with calculated kdiss and khydr constants (A) and quantification of the two constants over different concentrations of Dhit (B).
(C and D) BODIPY-GTPγS was used to probe the GDI activity of Dhit toward Gαo, showing an example of individual BODIPY-GTPγS uptake curves with calculated kdiss constants (C) and quantification of the initial BODIPY-GTPγS uptake rate over different concentrations of Dhit (D).
(E) The GDI activity of Dhit is also seen in the [35S]GTPγS assay; the resulting kdiss rate constants are shown (difference between the two kdiss values is statistically significant: p value < 0.05 by the Student’s t test).
(F) The GDI activity is resided in the RGS domain of Dhit; N-Dhit has no activity. Data are present as mean ± SEM (n ≥ 3). 1 μM Gαo was used in all panels. See also Figure S2.
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5. Figure 3
Crystal Structure of the RGS Domain of Dhit and Insights into the Molecular Mechanism of GDI Activity
(A and B) Structural alignment of the RGS domain of Dhit (blue) and RGS17 (orange, A) or RGS16 (magenta, B) reveals very similar folds.
(C) Model of the structure of mouse Gαo-GDP (with its Ras-like domain in green and helical domain in yellow) in complex with the RGS domain of RGS16 (magenta) superimposed with that of Dhit (blue).
(D) Magnified region nearby Arg242 of Dhit. Bonds between Arg242 (or Lys164 of RGS16) and the contact amino acid residues in Gαo are shown in blue (or red); distances are given in Å (distance between Lys164 of RGS16 and Asp116 of Gαo is not shown and is ∼4.8 Å).
(E) Alignment of the partial amino acid sequences of Drosophila Dhit and Loco and human RGS proteins of the RZ (19, 17, 20), R4 (16, 1), and R12 families (12, 14). Numbering of Dhit amino acids is shown above the sequence. Arg242 of Dhit and identical arginine residues of human RZ RGS proteins are in red. R4 family RGS proteins have lysine in this position (violet), and R12 members phenylalanine (green).
(F) The GDI activity of Dhit and Dhit[R242K] toward Drosophila Gαo. It can be seen that 20 μM Dhit[R242K] has GDI activity comparable to 5 μM Dhit wild-type.
(G) In pull-down experiments, Gαo-GDP efficiently precipitated both wild-type Dhit and Dhit[R242K]. See also Figures S2, S3, and S4.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Evidence for the GAP-Independent Activities of Dhit and RGS19 in Drosophila and HEK293T Cells and Model of the Double-Negative RGS Action
(A) Defects in composition of stout sensory bristles of the adult wing margin (red arrows) are infrequent upon RNAi-driven downregulation of dhit (upper panel), clearly visible upon expression of Gαo[Q205L] (middle panel), and stimulated upon co-expression of RNAi-dhit and Gαo[Q205L] (lower panel).
(B) Quantification (n ≥ 10 wings) of bristle defects induced by Gαo[Q205L] expressed in the RNAi-dhit or null dhit background (dhitex183/dhitex274 transheterozygous) confirms strong stimulation of Gαo[Q205L] activity by reduction in Dhit levels.
(C) Forskolin-stimulated CREB activation in HEK293T cells is suppressed by constitutively active Gαo and Gαi3—the action reverted by RGS19 or Dhit, but not by the RGS19[R190K] mutant form. Statistical significance in (B) and (C) was assessed by the Student’s t test; ∗∗∗ indicates p value < ; ∗∗, p < Data are present as mean ± SEM.
(D) Model of the double-negative action of Dhit and related RGS proteins. Ligand-bound GPCR dissociates the heterotrimeric Gαβγ complex, releasing Gβγ and Gα-GTP, both competent of signaling to downstream effectors. The intrinsic GTPase activity of Gα hydrolyzes GTP to GDP; the resultant Gα-GDP is prone to interact with Gβγ and cease the signaling cycle. Alternatively, it can re-exchange its nucleotide back to GTP and continue signaling. Dhit and related RGS proteins exert two negative effects on Gα: one speeds up GTP hydrolysis by and the other prevents reactivation of the Gα subunit, robustly restricting G protein activity. See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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