G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) consist of a nucleotide-binding alpha subunit and a high- affinity complex of beta and gamma subunits. There is molecular heterogeneity of beta and gamma, but the significance of this diversity is poorly understood. Different G protein beta and gamma subunits have been expressed both singly and in combinations in Sf9 cells. Although expression of individual subunits is achieved in all cases, beta gamma subunit activity (support of pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1) is detected only when beta and gamma are expressed concurrently. Of the six combinations of beta gamma tested (beta 1 or beta 2 with gamma 1, gamma 2, or gamma 3), only one, beta 2 gamma 1, failed to generate a functional complex. Each of the other five complexes has been purified by subunit exchange chromatography using Go alpha-agarose as the chromatographic matrix. We have detected differences in the abilities of the purified proteins to support ADP- ribosylation of Gi alpha 1; these differences are attributable to the gamma component of the complex. When assayed for their ability to inhibit calmodulin-stimulated type-I adenylylcyclase activity or to potentiate Gs alpha-stimulated type-II adenylylcyclase, recombinant beta 1 gamma 1 and transducin beta gamma are approximately 10 and 20 times less potent, respectively, than the other complexes examined. Prenylation and/or further carboxyl-terminal processing of gamma are not required for assembly of the beta gamma subunit complex but are indispensable for high affinity interactions of beta gamma with either G protein alpha subunits or adenylylcyclases

The structure of the G protein heterotrimer G\u3csub\u3eiα1\u3c/sub\u3eβ\u3csub\u3e1\u3c/sub\u3eγ\u3csub\u3e2\u3c/sub\u3e

The crystallographic structure of the G protein heterotrimer Giα1(GDP)β1γ2 (at 2.3 A) reveals two nonoverlapping regions of contact between α and β, an extended interface between β and nearly all of γ, and limited interaction of α with γ. The major α/β interface covers switch II of α, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with α-GDP) explain stabilization of the inactive conformation of α by βγ. Repeated WD motifs in β form a circularized sevenfold β propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade

The structure of the G protein heterotrimer Giα1β1γ2

AbstractThe crystallographic structure of the G protein heterotrimer Giα1(GDP)β1γ2 (at 2.3 A) reveals two nonoverlapping regions of contact between α and β, an extended interface between β and nearly all of γ, and limited interaction of α with γ. The major α/β interface covers switch II of α, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with α-GDP) explain stabilization of the inactive conformation of α by βγ. Repeated WD motifs in β form a circularized sevenfold β propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade

Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization. beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate. The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain. We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity. We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system. Gi alpha-mediated inhibition of adenylyl cyclase was not affected. These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain

RSUME enhances Glucocorticoid Receptor SUMOylation and transcriptional activity

Glucocorticoid receptor (GR) activity is modulated by posttranslational modifications, including phosphorylation, ubiquitination, and SUMOylation. The GR has three SUMOylation sites: lysine 297 (K297) and K313 in the N-terminal domain (NTD) and K721 within the ligand-binding domain. SUMOylation of the NTD sites mediates the negative effect of the synergy control motifs of GR on promoters with closely spaced GR binding sites. There is scarce evidence on the role of SUMO conjugation to K721 and its impact on GR transcriptional activity. We have previously shown that RSUME (RWD-containing SUMOylation enhancer) increases protein SUMOylation.We nowdemonstrate that RSUME interacts with the GR and increases its SUMOylation. RSUME regulatesGRtranscriptional activity and the expression of its endogenous target genes, FKBP51 and S100P. RSUME uncovers a positive role for the third SUMOylation site, K721, on GR-mediated transcription, demonstrating thatGRSUMOylation acts positively in the presence of a SUMOylation enhancer. Both mutation of K721 and small interfering RNA-mediated RSUME knockdown diminish GRIP1 coactivator activity. RSUME, whose expression is induced under stress conditions, is a key factor in heat shock-inducedGRSUMOylation. These results show that inhibitory and stimulatorySUMOsites are present in theGRand at higher SUMOylation levels the stimulatory one becomes dominant.Fil: Druker, Jimena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Liberman, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Antunica Noguerol, María de Las Nieves. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Gerez, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; ArgentinaFil: Paez Pereda, Marcelo. Max Planck Institute of Psychiatry; AlemaniaFil: Rein, Theo. Max Planck Institute of Psychiatry; AlemaniaFil: Iñiguez Lluhi, Jorge A.. University of Michigan Medical School. Department of Pharmacology; Estados UnidosFil: Holsboer, Florian. Max Planck Institute of Psychiatry; AlemaniaFil: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires; Argentin