Structural basis of activity and subunit recognition in G protein heterotrimers

Background: Inactive heterotrimeric G proteins are composed of a GDP-bound α subunit (G(α)) and a stable heterodimer of G(β) and G(γ) subunits. Upon stimulation by a receptor, G(α) subunits exchange GDP for GTP and dissociate from G(βγ) both G(α) and G(βγ) then interact with downstream effectors. Isoforms of G(α), G(β) and G(γ) potentially give rise to many heterotrimeric combinations, limited in part by amino acid sequence differences that lead to selective interactions. The mechanism by which GTP promotes G(βγ) dissociation is incompletely understood. The Gly203→Ala mutant of G(iα1) binds and hydrolyzes GTP normally but does not dissociate from G(βγ) demonstrating that GTP binding and activation can be uncoupled. Structural data are therefore important for understanding activation and subunit recognition in G protein heterotrimers. Results: The structures of the native (G(iα1β1γ2)) heterotrimer and that formed with Gly203→AlaG(iα1) have been determined to resolutions of 2.3 Å, and 2.4 Å, respectively, and reveal previously unobserved segments at the G(γ2) C terminus. The Gly203→Ala mutation alters the conformation of the N terminus of the switch II region (Val201-Ala203), but not the global structure of the heterotrimer. The N termini of G(β) and G(γ) form a rigid coiled coil that packs at varying angles against the β propeller of G(β). Conformational differences in the CD loop of β blade 2 of G(β) mediate isoform-specific contacts with G(α). Conclusions: The Gly203→Ala mutation in G(iα1) blocks the conformational changes in switch II that are required to release G(βγ) upon binding GTP. The interface between the ras-like domain of G, and the β propeller of G(β) appears to be conserved in all G protein heterotrimers. Sequence variation at the G(β)-G(α) interface between the N-terminal helix of G(α) and the CD loop of β blade 2 of G(β1) (residues 127-135) could mediate isoform-specific contacts. The specificity of G(β) and Gγ interactions is largely determined by sequence variation in the contact region between helix 2 of Gγ and the surface of G(β)

The A326S mutant of G(iα1) as an approximation of the receptor-bound state

Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their α subunits. In search of an approximation of the receptor-α subunit complex, we have considered the properties of A326S G(iα1), a mutation discovered originally in G(sα) (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissociation of GDP from the α(i1)β1γ2 heterotrimer by 250-fold. Nevertheless, affinity of mutant G(iα1) for GTPγS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the α subunit. The mutation also uncouples two activities of βγ: stabilization of the GDP-bound α subunit (which is retained) and retardation of GDP dissociation heterotrimer (which is lost). For wild-type and mutant G(iαl), βγ prevents irreversible inactivation of the α subunit at 30 °C. However, the mutation accelerates irreversible inactivation of α at 37 °C despite the presence of βγ. Structurally, the mutation weakens affinity for GTPγS by steric crowding: a 2-fold increase in the number of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those containing A326S G(i 1/2 ). However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers containing A326S G(iα1). In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissociation of GDP can be observed in the absence of substantial structural alteration of a G(α) subunit in the GDP-bound state

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

Compositions and Methods of Modulating 15-PGDH Activity

Compounds and methods of modulating 15-PGDH activity, modulating tissue prostaglandin levels, treating disease, diseases disorders, or conditions in which it is desired to modulate 15-PGDH activity and/or prostaglandin levels include 15-PGDH inhibitors and 15-PGDH activators described herein

Compositions and Methods of Modulating 15-PGDH Activity

Compounds and methods of modulating 15-PGDH activity, modulating tissue prostaglandin levels, treating disease, diseases disorders, or conditions in which it is desired to modulate 15-PGDH activity and/or prostaglandin levels include 15-PGDH inhibitors and 15-PGDH activators described herein

Ethnicity, voter alignment and political party affiliation - an African case: Zambia

Conventional wisdom holds that ethnicity provides the social cleavage for voting behav-iour and party affiliation in Africa. Because this is usually inferred from aggregate data of national election results, it might prove to be an ecological fallacy. The evidence based on individual data from an opinion survey in Zambia suggests that ethnicity matters for voter alignment and even more so for party affiliation, but it is certainly not the only factor. The analysis also points to a number of qualifications which are partly methodology-related. One is that the degree of ethnic voting can differ from one ethno-political group to the other depending on various degrees of ethnic mobilisation. Another is that if smaller eth-nic groups or subgroups do not identify with one particular party, it is difficult to find a significant statistical correlation between party affiliation and ethnicity - but that does not prove that they do not affiliate along ethnic lines.Wahlverhalten und Mitgliedschaft in politischen Parteien Afrikas ist nur wenig untersucht worden. Gewöhnlich wird argumentiert, dass Ethnizität als soziale Konfliktlinie das Wahlverhalten und die Parteienmitgliedschaft strukturiert. Da dieses Argument auf hoch aggregierten Wahldaten beruht, kann hier ein ökologischer Fehlschuss vorliegen. Die vorliegende Analyse beruht deshalb auf individuellen Umfragedaten aus Sambia. Das Ergebnis ist, dass Ethnizität tatsächlich eine Rolle für das Wahlverhalten und die Parteienmitgliedschaft spielt, aber keineswegs den einzigen Erklärungsfaktor darstellt. Die Analyse offenbart zudem eine Reihe von Einschränkungen und Qualifizierungen, die teilweise methodischer Natur sind. Eine ist, dass ethnisches Wahlverhalten und Parteienmitgliedschaft von einer ethnischen Gruppe zur anderen unterschiedlich ist, dass, wenn sich kleinere ethnische Gruppen oder Untergruppen mit keiner Partei identifizieren, es schwierig wird, statistisch signifikante Korrelationen zu finden - was indessen noch nicht beweist, dass Ethnizität keine Rolle spielt

Balance-of-Powers Arguments and the Structural Constitution

Balance-of-powers arguments are ubiquitous in judicial opinions and academic articles that address separation-of-powers disputes over the president\u27s removal authority, power to disregard statutes, authority to conduct foreign wars, and much else. However, the concept of the balance of powers has never received a satisfactory theoretical treatment. This Essay examines possible theories of the balance of powers and rejects them all as unworkable and normatively questionable. Judges and scholars should abandon the balance-of-powers metaphor and instead address directly whether bureaucratic innovation is likely to improve policy outcomes

Treatment for Adolescents with Depression Study (TADS): safety results.

OBJECTIVE: To compare the rates of physical, psychiatric, and suicide-related events in adolescents with MDD treated with fluoxetine alone (FLX), cognitive-behavioral therapy (CBT), combination treatment (COMB), or placebo (PBO). METHOD: Safety assessments included adverse events (AEs) collected by spontaneous report, as well as systematic measures for specific physical and psychiatric symptoms. Suicidal ideation and suicidal behavior were systematically assessed by self- and clinician reports. Suicidal events were also reanalyzed by the Columbia Group and expert raters using the Columbia-Classification Algorithm for Suicidal Assessment used in the U.S. Food and Drug Administration reclassification effort. RESULTS: Depressed adolescents reported high rates of physical symptoms at baseline, which improved as depression improved. Sedation, insomnia, vomiting, and upper abdominal pain occurred in at least 2% of those treated with FLX and/or COMB and at twice the rate of placebo. The rate of psychiatric AEs was 11% in FLX, 5.6% in COMB, 4.5% in PBO, and 0.9% in CBT. Suicidal ideation improved overall, with greatest improvement in COMB. Twenty-four suicide-related events occurred during the 12-week period: 5 patients (4.7%) in COMB, 10 (9.2%) in FLX, 5 (4.5%) in CBT, and 3 (2.7%) in placebo. Statistically, only FLX had more suicide-related events than PBO (p =.0402, odds ratio (OR) = 3.7, 95% CI 1.00-63.7). Only five actual attempts occurred (2 COMB, 2 FLX, 1 CBT, 0 PBO). There were no suicide completions. CONCLUSIONS: Different methods for eliciting AEs produce different results. In general, as depression improves, physical complaints and suicidal ideation decrease in proportion to treatment benefit. In this study, psychiatric AEs and suicide-related events are more common in FLX-treated patients. COMB treatment may offer a more favorable safety profile than medication alone in adolescent depression