Delayed internalization and lack of recycling in a beta₂-adrenergic receptor fused to the G protein alpha-subunit

<p>Abstract</p> <p>Background</p> <p>Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β₂-adrenergic receptor (β₂AR) and Gα_sindicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gα_s-fused β₂AR.</p> <p>Results</p> <p>The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization.</p> <p>Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β₂AR returned rapidly to the plasma membrane, β₂AR-Gα_sdid not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis.</p> <p>Conclusion</p> <p>The covalent linkage between β₂AR and Gα_sdoes not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes, but is an essential requirement for the correct post-endocytic sorting and recycling of the two proteins.</p

Reduced plasmodium parasite burden associates with CD38(+) CD4(+) T cells displaying cytolytic potential and impaired IFN-gamma production

Using a unique resource of samples from a controlled human malaria infection ( CHMI) study, we identified a novel population of CD4(+) T cells whose frequency in the peripheral blood was inversely correlated with parasite burden following P. falciparum infection. These CD4(+) T cells expressed the multifunctional ectoenzyme CD38 and had unique features that distinguished them from other CD4(+) T cells. Specifically, their phenotype was associated with proliferation, activation and cytotoxic potential as well as significantly impaired production of IFN-gamma and other cytokines and reduced basal levels of activated STAT1. A CD38(+) CD4(+) T cell population with similar features was identified in healthy uninfected individuals, at lower frequency. CD38(+) CD4(+) T cells could be generated in vitro from CD38(-) CD4(+) T cells after antigenic or mitogenic stimulation. This is the first report of a population of CD38(+) CD4(+) T cells with a cytotoxic phenotype and markedly impaired IFN-gamma capacity in humans. The expansion of this CD38(+) CD4(+) T population following infection and its significant association with reduced blood-stage parasite burden is consistent with an important functional role for these cells in protective immunity to malaria in humans. Their ubiquitous presence in humans suggests that they may have a broad role in host-pathogen defense

Bone-Breaking Bite Force of Basilosaurus isis (Mammalia, Cetacea) from the Late Eocene of Egypt Estimated by Finite Element Analysis

Bite marks suggest that the late Eocence archaeocete whale Basilosaurus isis (Birket Qarun Formation, Egypt) fed upon juveniles of the contemporary basilosaurid Dorudon atrox. Finite element analysis (FEA) of a nearly complete adult cranium of B. isis enables estimates of its bite force and tests the animal’s capabilities for crushing bone. Two loadcases reflect different biting scenarios: 1) an intitial closing phase, with all adductors active and a full condylar reaction force; and 2) a shearing phase, with the posterior temporalis active and minimized condylar force. The latter is considered probable when the jaws were nearly closed because the preserved jaws do not articulate as the molariform teeth come into occulusion. Reaction forces with all muscles active indicate that B. isis maintained relatively greater bite force anteriorly than seen in large crocodilians, and exerted a maximum bite force of at least 16,400 N at its upper P3. Under the shearing scenario with minimized condylar forces, tooth reaction forces could exceed 20,000 N despite lower magnitudes of muscle force. These bite forces at the teeth are consistent with bone indentations on Dorudon crania, reatract-and-shear hypotheses of Basilosaurus bite function, and seizure of prey by anterior teeth as proposed for other archaeocetes. The whale’s bite forces match those estimated for pliosaurus when skull lengths are equalized, suggesting similar tradeoffs of bite function and hydrodynamics. Reaction forces in B. isis were lower than maxima estimated for large crocodylians and carnivorous dinosaurs. However, comparison of force estimates from FEA and regression data indicate that B. isis exerted the largest bite forces yet estimated for any mammal, and greater force than expected from its skull width. Cephalic feeding biomechanics of Basilosaurus isis are thus consistent with habitual predation