Conformational changes in the G protein Gs induced by the β2 adrenergic receptor

G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β(2) adrenergic receptor (β(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the β(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the β(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.     

Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins

Understanding the energetics of molecular interactions is fundamental to all of the central quests of structural biology including structure prediction and design, mapping evolutionary pathways, learning how mutations cause disease, drug design, and relating structure to function. Hydrogen-bonding is widely regarded as an important force in a membrane environment because of the low dielectric constant of membranes and a lack of competition from water. Indeed, polar residue substitutions are the most common disease-causing mutations in membrane proteins. Because of limited structural information and technical challenges, however, there have been few quantitative tests of hydrogen-bond strength in the context of large membrane proteins. Here we show, by using a double-mutant cycle analysis, that the average contribution of eight interhelical side-chain hydrogen-bonding interactions throughout bacteriorhodopsin is only 0.6 kcal mol(-1). In agreement with these experiments, we find that 4% of polar atoms in the non-polar core regions of membrane proteins have no hydrogen-bond partner and the lengths of buried hydrogen bonds in soluble proteins and membrane protein transmembrane regions are statistically identical. Our results indicate that most hydrogen-bond interactions in membrane proteins are only modestly stabilizing. Weak hydrogen-bonding should be reflected in considerations of membrane protein folding, dynamics, design, evolution and function.     

APJ acts as a dual receptor in cardiac hypertrophy

Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of β-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.     

Superallowed Gamow-Teller decay of the doubly magic nucleus 100Sn

The shell structure of atomic nuclei is associated with 'magic numbers' and originates in the nearly independent motion of neutrons and protons in a mean potential generated by all nucleons. During β(+)-decay, a proton transforms into a neutron in a previously not fully occupied orbital, emitting a positron-neutrino pair with either parallel or antiparallel spins, in a Gamow-Teller or Fermi transition, respectively. The transition probability, or strength, of a Gamow-Teller transition depends sensitively on the underlying shell structure and is usually distributed among many states in the neighbouring nucleus. Here we report measurements of the half-life and decay energy for the decay of (100)Sn, the heaviest doubly magic nucleus with equal numbers of protons and neutrons. In the β-decay of (100)Sn, a large fraction of the strength is observable because of the large decay energy. We determine the largest Gamow-Teller strength so far measured in allowed nuclear β-decay, establishing the 'superallowed' nature of this Gamow-Teller transition. The large strength and the low-energy states in the daughter nucleus, (100)In, are well reproduced by modern, large-scale shell model calculations.     

Gene therapy: therapeutic gene causing lymphoma

The development of T-cell leukaemia following the otherwise successful treatment of three patients with X-linked severe combined immune deficiency (X-SCID) in gene-therapy trials using haematopoietic stem cells has led to a re-evaluation of this approach. Using a mouse model for gene therapy of X-SCID, we find that the corrective therapeutic gene IL2RG itself can act as a contributor to the genesis of T-cell lymphomas, with one-third of animals being affected. Gene-therapy trials for X-SCID, which have been based on the assumption that IL2RG is minimally oncogenic, may therefore pose some risk to patients.     

The role of volatiles in magma chamber dynamics

Many andesitic volcanoes exhibit effusive eruption activity, with magma volumes as large as 10(7)-10(9) m(3) erupted at rates of 1-10 m(3) x s(-1) over periods of years or decades. During such eruptions, many complex cycles in eruption rates have been observed, with periods ranging from hours to years. Longer-term trends have also been observed, and are thought to be associated with the continuing recharge of magma from deep in the crust and with waning of overpressure in the magma reservoir. Here we present a model which incorporates effects due to compressibility of gas in magma. We show that the eruption duration and volume of erupted magma may increase by up to two orders of magnitude if the stored internal energy associated with dissolved volatiles can be released into the magma chamber. This mechanism would be favoured in shallow chambers or volatile-rich magmas and the cooling of magma by country rock may enhance this release of energy, leading to substantial increases in eruption rate and duration.     

Repetitive transient rises in cytoplasmic free calcium in hormone-stimulated hepatocytes

In the stressed animal, the vasoactive hormones vasopressin and angiotensin-II and the neurotransmitter noradrenaline induce liver cells to release glucose from glycogen. The intracellular signal that links the cell-surface receptors for noradrenaline (alpha 1) and vasoactive peptides to activation of glycogenolysis is known to be a rise in the cytoplasmic concentration of free calcium ions (free Ca). The receptors for these agonists induce the hydrolysis of phosphatidylinositol 4,5-bisphosphate, a minor plasmalemma lipid, to produce inositol trisphosphate and diacylglycerol. Inositol trisphosphate has been shown to mobilize intracellular calcium in hepatocytes. We show here, by means of aequorin measurements in single, isolated rat hepatocytes, that the free Ca response to these agonists consists of a series of transients. Each transient rose within 3 s to a peak free Ca of at least 600 nM and had a duration of approximately 7 s. The transients were repeated at intervals of 0.3-4 min, depending on agonist concentration. Between transients, free Ca returned to the resting level of approximately 200 nM. Clearly, the mechanisms controlling free Ca in hepatocytes are more complex than hitherto suspected.     

An expanding radio nebula produced by a giant flare from the magnetar SGR 1806-20

Soft gamma-ray repeaters (SGRs) are 'magnetars', a small class of slowly spinning neutron stars with extreme surface magnetic fields, B approximately 10(15) gauss (refs 1 , 2 -3). On 27 December 2004, a giant flare was detected from the magnetar SGR 1806-20 (ref. 2), only the third such event recorded. This burst of energy was detected by a variety of instruments and even caused an ionospheric disturbance in the Earth's upper atmosphere that was recorded around the globe. Here we report the detection of a fading radio afterglow produced by this outburst, with a luminosity 500 times larger than the only other detection of a similar source. From day 6 to day 19 after the flare from SGR 1806-20, a resolved, linearly polarized, radio nebula was seen, expanding at approximately a quarter of the speed of light. To create this nebula, at least 4 x 10(43) ergs of energy must have been emitted by the giant flare in the form of magnetic fields and relativistic particles.     

A splicing mutation affecting expression of ataxia-telangiectasia and Rad3-related protein (ATR) results in Seckel syndrome

Seckel syndrome (OMIM 210600) is an autosomal recessive disorder characterized by intrauterine growth retardation, dwarfism, microcephaly and mental retardation. Clinically, Seckel syndrome shares features in common with disorders involving impaired DNA-damage responses, such as Nijmegen breakage syndrome (OMIM 251260) and LIG4 syndrome (OMIM 606593). We previously mapped a locus associated with Seckel syndrome to chromosome 3q22.1-q24 in two consanguineous Pakistani families. Further marker analysis in the families, including a recently born unaffected child with a recombination in the critical region, narrowed the region to an interval of 5 Mbp between markers D3S1316 and D3S1557 (145.29 Mbp and 150.37 Mbp). The gene encoding ataxia-telangiectasia and Rad3-related protein (ATR) maps to this region. A fibroblast cell line derived from an affected individual displays a defective DNA damage response caused by impaired ATR function. We identified a synonymous mutation in affected individuals that alters ATR splicing. The mutation confers a phenotype including marked microcephaly (head circumference 12 s.d. below the mean) and dwarfism (5 s.d. below the mean). Our analysis shows that UV-induced ATR activation can occur in non-replicating cells following processing by nucleotide excision repair.