Ric-8B stabilizes the alpha subunit of stimulatory G protein by inhibiting its ubiquitination

Abstract

The alpha subunit of stimulatory G protein (G alpha(s)) activates adenylyl cyclase, which catalyzes cAMP production, and regulates many physiological aspects, such as cardiac regulation and endocrine systems. Ric-8B (resistance to inhibitors of cholinesterase 8B) has been identified as the G alpha(s)-binding protein; however, its role in G(s) signaling remains obscure. In this study, we present evidence that Ric-8B specifically and positively regulates G(s) signaling by stabilizing the G alpha(s) protein. An in vitro biochemical study suggested that Ric-8B does not possess guanine nucleotide exchange factor activity. However, knockdown of Ric-8B attenuated beta-adrenergic agonist-induced cAMP accumulation, indicating that Ric-8B positively regulates G(s) signaling. Interestingly, overexpression and knockdown of Ric-8B resulted in an increase and a decrease in the G alpha(s) protein, respectively, without affecting the G alpha(s) mRNA level. We found that the G alpha(s) protein is ubiquitinated and that this ubiquitination is inhibited by Ric-8B. This Ric-8B-mediated inhibition of G alpha(s) ubiquitination requires interaction between Ric-8B and G alpha(s) because Ric-8B splicing variants, which are defective for G alpha(s) binding, failed to inhibit the ubiquitination. Taken together, these results suggest that Ric-8B plays a critical and specific role in the control of G alpha(s) protein levels by modulating G alpha(s) ubiquitination and positively regulates G(s) signaling.

FIGURE 1.

Effect of Ric-8B on guanine nucleotide exchange. A, GST or GST-Ric-8B (100 nm each) was incubated with Gα_s or Gα_q (100 nm each) for 1 h at 25 °C. These mixtures were bound to glutathione-Sepharose and washed extensively with a buffer. The proteins were eluted with an SDS-PAGE sample buffer. Co-precipitation of Gα proteins was detected by immunoblotting (IB). B and C, Gα_q (B) and Gα_s (C) (5 pmol each) were incubated with [³⁵S]GTPγS in reaction mixture (100 μl) containing GST (●), GST-Ric-8B (▲), or GST-Ric-8A (▼) (20 pmol each). Aliquots (20 μl) of these reaction mixtures were taken at the indicated time points and filtered to absorb nucleotide-bound protein. The amount of G protein-bound [³⁵S]GTPγS was determined by scintillation counting.

FIGURE 2.

Knockdown of Ric-8B decreases isoproterenol-induced cAMP accumulation. A, cell lysates from NIH3T3 cells infected with retroviruses encoding control shRNA or two different sequences of shRNAs directed against Ric-8B were analyzed by immunoblotting (IB) using anti-Ric-8B and anti-tubulin antibodies. B, shown is the time course of cAMP accumulation. cAMP accumulation of NIH3T3 cells infected with retroviruses encoding control (○) or Ric-8B2 (shRic-8B#2; ●) shRNA was measured following exposure to 10 μm isoproterenol for the indicated times. C, NIH3T3 cells infected with retroviruses were exposed to 10 μm isoproterenol for 20 min, and cAMP accumulation was measured. The data are expressed as the mean ± S.D. from three independent experiments. CTL, control.

FIGURE 3.

Ric-8B positively regulates Gα_s protein levels. A, cell lysates were prepared from NIH3T3 cells infected with retroviruses encoding control shRNA or two different sequences of shRNAs directed against Ric-8B (shRic-8B) and were analyzed by immunoblotting (IB) using the indicated antibodies. The Gα_s protein exists as two spliced forms, short and long. The ratio of the spliced forms varies in different cell types. The positions of the long and short Gα_s variants are indicated by lines. B, HEK293T cells were transfected with FLAG-Ric-8B and harvested for RNA and protein preparations. Semiquantitative reverse transcription-PCR (RT-PCR) and immunoblotting were performed to assess the effect of Ric-8B on the Gα_s mRNA and protein levels, respectively. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 4.

Ric-8B inhibits Gα_s degradation and ubiquitination. A, HEK293T cells were treated with 10 μm MG132 for 12 h, and cell lysates were analyzed by immunoblotting (IB) using anti-Gα_s and anti-actin antibodies. B, HEK293T cells were pre-incubated with 10 μm MG132 for 8 h, and cAMP accumulation was then measured following exposure to 10 nm pituitary adenylate cyclase-activating polypeptide (PACAP) for 15 min. C, HEK293T cells transfected with an empty vector or FLAG-Ric-8B were treated with 100 μg/ml CHX for the indicated times. Cell lysates were analyzed by immunoblotting using anti-Gα_s, anti-FLAG, and anti-actin antibodies. D, HEK293T cells were transfected with Gα_s-short, His-ubiquitin (His-Ub), and FLAG-Ric-8B. Twenty-four hours after transfection, cells were treated with 10 μm MG132 for 12 h and subsequently lysed with an 8 m urea-containing buffer, and His-tagged ubiquitinated proteins were precipitated with nickel-agarose resin. Ubiquitinated Gα_s was detected by immunoblotting using the anti-Gα_s antibody.

FIGURE 5.

Interaction of Ric-8B with Gα_s is important for inhibiting Gα_s ubiquitination. HEK293T cells were transfected with Gα_s-short, His-ubiquitin (His-Ub), and FLAG-tagged full-length Ric-8B (FL) or spliced variants (A) or with His-ubiquitin, FLAG-Ric-8B, and wild-type Gα_s-short (Gα_sWT) or Gα_s/i-SWII_i (B). The ubiquitinated Gα_s was detected as described in the legend to Fig. 4D.