Effect of pathogenic cysteine mutations on FGFR3 transmembrane domain dimerization in detergents and lipid bilayers

Mutations in fibroblast growth factor receptors are known as the genetic basis of skeletal growth disorders. The mechanism of pathogenesis, as determined by mutation-induced changes in receptor structure, interactions, and function, is elusive. Here we study three pathogenic Cys mutations, associated with either thanatophoric dysplasia or achondroplasia, in the TM domain of fibroblast growth factor receptors 3 (FGFR3). We characterize the dimerization propensities of the mutant TM domains in detergents and in lipid bilayers, in the presence and absence of reducing agents, and compare them to previous measurements of wild-type. We find that the Cys mutations increase the propensity for dimerization in detergent, with the Cys370 mutant exhibiting the highest propensity for disulfide bond formation, the Cys371 mutant having an intermediate propensity, and Cys375 the lowest. Thus, disulfide bonds readily form in detergents, with efficiency that correlates with the severity of the phenotype. In lipid bilayers, however, the Cys370 mutant, which dimerizes strongly in detergent, behaves as the wild-type, suggesting that Cys370-mediated disulfide bonds do not form between the isolated TM domains in bilayers. Thus, the nature of the hydrophobic environment plays an important role in defining the structure and flexibility of transmembrane dimers. These results and previous findings from cellular studies lead us to propose a conformational flexibility mechanism of receptor stabilization as a basis for disregulated FGFR3 signaling in thanatophoric dysplasia and achondroplasia.     

A first insight into population structure and linkage disequilibrium in the US peanut minicore collection

Knowledge of genetic diversity, population structure, and degree of linkage disequilibrium (LD) in target association mapping populations is of great importance and is a prerequisite for LD-based mapping. In the present study, 96 genotypes comprising 92 accessions of the US peanut minicore collection, a component line of the tetraploid variety Florunner, diploid progenitors A. duranensis (AA) and A. ipaënsis (BB), and synthetic amphidiploid accession TxAG-6 were investigated with 392 simple sequence repeat (SSR) marker bands amplified using 32 highly-polymorphic SSR primer pairs. Both distance- and model-based (Bayesian) cluster analysis revealed the presence of structured diversity. In general, the wild-species accessions and the synthetic amphidiploid grouped separately from most minicore accessions except for COC155, and were eliminated from most subsequent analyses. UPGMA analysis divided the population into four subgroups, two major subgroups representing subspecies fastigiata and hypogaea, a third group containing individuals from each subspecies or possibly of mixed ancestry, and a fourth group, either consisting of COC155 alone if wild species were excluded, or of COC155, the diploid species, and the synthetic amphidiploid. Model-based clustering identified four subgroups- one each for fastigiata and hypogaea subspecies, a third consisting of individuals of both subspecies or of mixed ancestry predominantly from Africa or Asia, and a fourth group, consisting of individuals predominantly of var fastigiata, peruviana, and aequatoriana accessions from South America, including COC155. Analysis of molecular variance (AMOVA) revealed statistically-significant (P < 0.0001) genetic variance of 16.87% among subgroups. A total of 4.85% of SSR marker pairs revealed significant LD (at r² ≥ 0.1). Of the syntenic marker pairs separated by distances < 10 cM, 11–20 cM, 21–50 cM, and > 50 cM, 19.33, 5.19, 6.25 and 5.29% of marker pairs were found in strong LD (P ≤ 0.01), in accord with LD extending to great distances in self pollinated crops. A threshold value of r² > 0.035 was found to distinguish mean r² values of linkage distance groups statistically from the mean r² values of unlinked markers; LD was found to extend to 10 cM over the entire minicore collection by this criterion. However, there were large differences in r² values among marker pairs even among tightly-linked markers. The implications of these findings with regard to the possibility of using association mapping for detection of genome-wide SSR marker-phenotype association are discussed.     

Insulin-induced beta-arrestin1 Ser-412 phosphorylation is a mechanism for desensitization of ERK activation by Galphai-coupled receptors

Beta-arrestin1 is an adapter/scaffold for many G protein-coupled receptors during mitogen-activated protein kinase signaling. Phosphorylation of beta-arrestin1 at position Ser-412 is a regulator of beta-arrestin1 function, and in the present study, we showed that insulin led to a time- and dose-dependent increase in beta-arrestin1 Ser-412 phosphorylation, which blocked isoproterenol- and lysophosphatidic acid-induced Ser-412 dephosphorylation and impaired ERK signaling by these G protein-coupled receptor ligands. Insulin treatment also led to accumulation of Ser-412-phosphorylated beta-arrestin1 at the insulin-like growth factor 1 receptor and prevented insulin-like growth factor 1/Src association. Insulin-induced Ser-412 phosphorylation was partially dependent on ERK as treatment with the MEK inhibitor PD98059 inhibited the insulin effect (62% reduction, p = 0.03). Inhibition of phosphatidylinositol 3-kinase by wortmannin did not have a significant effect (9% reduction, p = 0.41). We also found that the protein phosphatase 2A (PP2A) was in a molecular complex with beta-arrestin1 and that the PP2A inhibitor okadaic acid increased Ser-412 phosphorylation. Concomitant addition of insulin and okadaic acid did not produce an additive effect on Ser-412 phosphorylation, suggesting a common mechanism. Small t antigen specifically inhibited PP2A, and in HIRcB cells expressing small t antigen, beta-arrestin1 Ser-412 phosphorylation was increased, and insulin had no further effect. Insulin treatment caused increased beta-arrestin1 Ser-412 phosphorylation, which blocked mitogen-activated protein kinase signaling and internalization by beta-arrestin1-dependent receptors with no effect on beta-adrenergic receptor Gs-mediated cAMP production. These findings provide a new mechanism for insulin-induced desensitization of ERK activation by Galphai-coupled receptors.     

Mechanism of activation of human c-KIT kinase by internal tandem duplications of the juxtamembrane domain and point mutations at aspartic acid 816

The patients suffering from acidosis usually sign psychological deficits. The cerebral dysfunction is reportedly caused by an acid-induced functional impairment of GABAergic neurons; however, the role of pyramidal neurons in this process remains unclear. By using electrophysiological method and changing extracellular pH, we investigated the influence of acidic environment on pyramidal neurons in the cortical slices, such as their ability of firing spikes and response to synaptic inputs. A low pH of artificial cerebral spinal fluid elevates the responses of pyramidal neurons to excitatory synaptic inputs and their ability of encoding digital spikes, as well as reduces the signal transmission at GABAergic synapses. The elevated ability of neuronal spiking is associated with the decreases of refractory periods and threshold potentials. Therefore, acidosis deteriorates brain functions through making the activities between cortical pyramidal neurons and GABAergic neurons imbalanced toward the overexcitation of neural networks, a process similar to neural excitotoxicity.     

The superposition principle applied to grasping an object producing moments outside anatomically-defined axes

The superposition principle suggests that motor commands can be divided into individually controlled components that summate to produce complex motor actions. Previous studies have examined the validity of this principle in human grasping by changing moments acting on an object about a single anatomically-defined axis and asking subjects to hold the object while their forearm was constrained. Superposition was reflected as separate control of the grip force and moments required to prevent object slip and maintain orientation. The objective of this study was to examine the robustness of this principle by: 1) expanding the range of tasks to include those where moments act on an object with respect to moment arms not necessarily in line with the anatomically-defined axes; 2) asking subjects to hold the object in an unconstrained manner. Ten subjects were asked to lift and hold an object vertically under eighteen moment conditions. Force and moment data from all digits were analysed using principal components analysis (PCA). Different PCAs were run for variable sets corresponding to moments about the long axis of the forearm (M(x)), the vertical (M(y)) and grip (M(z)) axes, and for the entire dataset (M(3D)). The PCA showed grip force and moment variables on separate PCs for the M(x), M(y), and M(3D) variable sets. The M(3D) PCA also showed a separation of variables corresponding to moments about each anatomically-defined axis. Thus, the present results show that the superposition principle holds during natural manipulation of an object experiencing external moments outside the anatomically-defined axes.     

Connectivity of Caribbean coral populations: complementary insights from empirical and modelled gene flow

Understanding patterns of connectivity among populations of marine organisms is essential for the development of realistic, spatially explicit models of population dynamics. Two approaches, empirical genetic patterns and oceanographic dispersal modelling, have been used to estimate levels of evolutionary connectivity among marine populations but rarely have their potentially complementary insights been combined. Here, a spatially realistic Lagrangian model of larval dispersal and a theoretical genetic model are integrated with the most extensive study of gene flow in a Caribbean marine organism. The 871 genets collected from 26 sites spread over the wider Caribbean subsampled 45.8% of the 1900 potential unique genets in the model. At a coarse scale, significant consensus between modelled estimates of genetic structure and empirical genetic data for populations of the reef-building coral Montastraea annularis is observed. However, modelled and empirical data differ in their estimates of connectivity among northern Mesoamerican reefs indicating that processes other than dispersal may dominate here. Further, the geographic location and porosity of the previously described east-west barrier to gene flow in the Caribbean is refined. A multi-prong approach, integrating genetic data and spatially realistic models of larval dispersal and genetic projection, provides complementary insights into the processes underpinning population connectivity in marine invertebrates on evolutionary timescales.     

The metabolism of formyl-substituted benzanthracenes to hydroxymethyl metabolites in rat liver in vitro and in vivo

Hydroxylation of benzylic methyl carbon atoms on drugs and carcinogenic polycyclic aromatic hydrocarbons (PAHs) forms benzylic alcohols. Many carcinogenic and mutagenic PAHs bear a primary or secondary benzylic hydroxyl group attached to the meso-region of the molecule. According to the unified theory, PAHs bearing a benzylic hydroxyl group are proximate carcinogenic metabolites. This paper demonstrates that carcinogenic benz[a]anthracenes bearing a formyl group at the meso-region undergo enzymatic reductive metabolism to the corresponding carcinogenic benzylic alcohol in vitro and in vivo. The unified theory would then predict sulfuric acid esterification of such benzylic alcohols as the final common step in their metabolic activation to generate ultimate electrophilic benzylic carbocations. Finally, oxidative metabolism of 7-formylbenz[a]anthracenes gives rise to corresponding carboxylic acids and other oxygenated metabolites that are carcinogenically inert. Thus, oxidative metabolism of meso-region formyl compounds represents an avenue for the elimination of the carcinogen in a detoxified form.