Ric-8B stabilizes the alpha subunit of stimulatory G protein by inhibiting its ubiquitination.
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.
Department of Cell Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
Yusuke
Y
Nagai
Akiyuki
A
Nishimura
Kenji
K
Tago
Norikazu
N
Mizuno
Hiroshi
H
Itoh
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry; D054875:Ubiquitination
D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; C485884:Ric8a protein, mouse; C559967:Ric8b protein, mouse; D025801:Ubiquitin; D003513:Cycloheximide; D000242:Cyclic AMP; D044767:Ubiquitin-Protein Ligases; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs; C549039:RIC8B protein, human