Surveying nonvisual arrestins reveals allosteric interactions between functional sites

Arrestins are important scaffolding proteins that are expressed in all vertebrate animals. They regulate cell-signaling events upon binding to active G-protein coupled receptors (GPCR) and trigger endocytosis of active GPCRs. While many of the functional sites on arrestins have been characterized, the question of how these sites interact is unanswered. We used anisotropic network modeling (ANM) together with our covariance compliment techniques to survey all the available structures of the nonvisual arrestins to map how structural changes and protein-binding affect their structural dynamics. We found that activation and clathrin binding have a marked effect on arrestin dynamics, and that these dynamics changes are localized to a small number of distant functional sites. These sites include α-helix 1, the lariat loop, nuclear localization domain, and the C-domain β-sheets on the C-loop side. Our techniques suggest that clathrin binding and/or GPCR activation of arrestin perturb the dynamics of these sites independent of structural changes.     

Functional Role of Amino-Terminal Serine16 and Serine27 of Gαz in Receptor and Effector Coupling

Abstract: The α subunit of G_z (α_z) harbors two N-terminal serine residues (at positions 16 and 27) that serve as protein kinase C-mediated phosphorylation sites. The cognate residues in the α subunit of G_t1 provide binding surfaces for the β₁ subunit. We used three serine-to-alanine mutants of α_z to investigate the functional importance of the two N-terminal serine residues. Wild-type or mutant α_z was transiently coexpressed with different receptors and adenylyl cyclase isozymes in human embryonic kidney 293 cells, and agonist-dependent regulation of cyclic AMP accumulation was examined in a setting where all endogenous α subunits of G_i were inactivated by pertussis toxin. Replacement of one or both serine residues by alanine did not alter the ability of α_z to interact with δ-opioid, dopamine D₂, or adenosine A₁ receptors. Its capacity to inhibit endogenous and type VI adenylyl cyclases was also unaffected. Functional release of βγ subunits from the mutant α_z subunits was not impaired because they transduced βγ-mediated stimulation of type II adenylyl cyclase. Constitutively active mutants of all four α_z subunits were constructed by the introduction of a Q205L mutation. The activated mutants showed differential abilities to inhibit human choriogonadotropin-mediated cyclic AMP accumulation in luteinizing hormone receptor-transfected cells. Loss of both serine residues, but not either one alone, impaired the receptor-independent inhibition of adenylyl cyclase by the GTPase-deficient mutant. Thus, replacement of the amino-terminal serine residues of α_z has no apparent effect on receptor-mediated responses, but these serine residues may be essential for ensuring transition of α_z into the active conformation.     

Postcranial paleoneurology of the Diapsida

The relationships between the size of the spinal cord and the size of the neural canal, and between gross spinal cord anatomy and locomotor style, were documented in a wide range of living diapsids. Observed relationships were used to make predictions about spinal cord anatomy and about limb use and position in related fossil taxa. In particular, the data suggest that the brachial plexus, and therefore the cervical/dorsal vertebral transition, of the theropod dinosaurs studied was located considerably posterior to its presently accepted location, and that the forelimbs of the giant carnosaurs Tyrannosaurus rex and Carnotaurus sastrei were of biologically insignificant use. Neural canal measurements support previous interpretations of locomotor style in extinct crocodilians, and can be used to predict limb placement in plesiosaurs.     

Responsiveness of the lamb ductus arteriosus to prostaglandins and their metabolites

The relative potencies of the prostaglandins A₁, A₂, E₁, E₂, F_2α and their 15-keto-, 15-keto-13,14-dihydro-, and 13,14-dihydro-metabolites were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂. All the prostaglandins (except PGF_2α and its 15-keto-metabolites) relaxed the tissue. However, only PGE₁, E₂, and their 13,14-dihydro-metabolites, were effective at concentrations below 10⁻⁸ M. Therefore, events that alter metabolism of circulating PGs in the perinatal period may have significant effects on the relative patency or closure of the ductus arteriosus.     

Maternal nutrition in pregnancy. Part I: a review.

Maternal undernutrition may result in a greater deprivation of the fetus than has previously been believed. The infant not only may be "light for dates" but also has an increased risk of perinatal disability or death secondary to gross neurologic and developmental abnormalities. This article reviews current knowledge of the energy, protein, iron, vitamin, sodium and calcium requirements in pregnancy, with special reference to the management of the underweight and overweight pregnant women.     

Biosynthesis and metabolism of prostaglandins in chick epiphyseal cartilage.

Microsomal fractions of cells isolated from chick epiphyseal cartilage catalyzed the synthesis of prostaglandins from radiolabeled delta8,11,14-eicosatrienoic and from arachidonic acids. In addition, the microsomal supernatants contained both 15-hydroxyprostaglandin dehydrogenase and prostaglandin 15-keto delta13,14-reductase activities. Two major classes of prostaglandins (E and F) were synthesized; however, a major product which chromatographically behaves as PGA was also isolated. Synthetase activities were analyzed for pH optima and response to known stimulators and inhibitors of prostaglandin systhesis. The different activators had varying stimulatory effects on prostaglandin synthesis; the anti-inflammatory drugs were all strongly inhibitory. Synthetase activity in the growth plate was highest in the zone of hypertrophy, declining substantially in the more heavily calcified regions. Degradative enzyme activities were highest in the zone of maturation and significantly lower in the adjacent hypertrophic zone. The net effect of these opposing activities would be to elevate prostaglandin levels at the zone of hypertrophy, a finding which suggests that prostaglandins may play a role in the modulation of epiphyseal cartilage metabolism.     

Fermentation of L-aspartate by a saccharolytic strain of Bacteroides melaninogenicus.

Resting cells of Bacteroides melaninogenicus fermented L-[14C]aspartate as a single substrate. The 14C-labeled products included succinate, acetate, CO2, oxaloacetate, formate, malate, glycine, alanine, and fumarate in the relative percentages 68, 15, 9.9, 2.7, 1.8, 1.0, 0.7, 0.5, and 0.06, respectively, based on the total counts per minute of the L-[14C]aspartate fermented. Ammonia was produced in high amounts, indicating that 96% of the L-aspartate fermented was deaminated. These data suggest that L-aspartate is mainly being reduced through a number of intermediate reactions involving enzymes of the tricarboxylic acid cycle to succinate. L-[14C]asparagine was also fermented by resting cells of B. melaninogenicus to form L-aspartate, which was subsequently, but less actively, fermented.     

An inhibitor of dopamine uptake,LR5182, CIS-3-(3,4-dichlorophenyl)-2-N,N-dimethylaminomethyl-bicyclo-[2,2,2]-octane,hydrochloride

LR5182 inhibited the uptake of dopamine in rat striatal synaptosomes and the uptake of norepinephrine in cortical synaptosomes with inhibitor constants, Ki values, of 3nM and 58nM, respectively. It was only a week inhibitor of serotonin uptake in cortical synaptosomes with a Ki value of 1.7μM. The uptake of dopamine and norepinephrine were significantly lowered within an hour after an intraperitoneal injection of LR5182. Among known inhibitors of dopamine uptake in synaptosomes of rat brain, LR5182 is most effective and selective. The rigid structure of LR5182 (Figure 1) suggested a gauche conformation of dopamine to be favored by the striatal uptake of dopamine.     

Characterization by saturation studies of the in vivo binding of [3H]flunitrazepam in mouse brain

The in vivo binding of [3H]flunitrazepam [( 3H]Fln) was characterized in seven regions of the mouse brain. The binding showed saturability and linear Scatchard plots. Hill coefficients were close to unity. Data fitting to a hyperbola by least squares yielded consistent Kd values for all regions studied (0.36-0.6 pmol/mg protein). Bmax values ranged from 0.14 to 0.89 pmol/mg protein, a sixfold regional variation. The order of binding is as follows: cortex greater than hippocampus greater than midbrain = thalamus/hypothalamus greater than striatum much greater than cerebellum greater than brainstem, consistent with that obtained by in vitro binding. The in vivo receptor density and affinity are apparently lower in comparison with in vitro parameters. This is consistent with the observation that the Kd increases and Bmax decreases in vitro when the incubation temperature is increased from 0 degrees C. Non-specific binding has been estimated by displacement of in vivo binding by unlabelled ligand in vitro as well as by pretreatment with unlabelled ligand. The two alternative methods were compared and evaluated. It is concluded that the displacement method provides more reliable estimates of the nonspecific binding. Diazepam-sensitive mice did not differ from the control mice in the in vivo [3H]Fln binding. However, mice pretreated with diazepam 1 or 2 days before have binding reduced by 70 or 30%, respectively. The reduced binding may be explained by receptor occupancy by residual oxazepam. However, the low concentration of the residual oxazepam is an unlikely cause of the phenomenon of "acute tolerance" observed in these mice.     

Identification of a membrane-associated 1-0-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine hydrolyzing phospholipase A2 in guinea pig 1 epidermis. Implications in the cutaneous biosynthesis of platelet-activating factor.

A membrane-associated 1-0-alkyl-2-arachidonoyl-GPC hydrolyzing phospholipase A2 was identified in guinea pig epidermis. It is regio-specific (associated with the particulate microsomal fraction) and specific for the hydrolysis of 1-0-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine. It is sensitive to low calcium concentrations suggesting that it may be activated by increasing intracellular calcium. Since ether-linked phospholipids are known to exist in the epidermis, further understanding of the properties of this 1-0-alkyl-arachidonoyl-hydrolyzing PLA2 may allow us to control the generation of 1-0-alkyl-2-lyso-sn-glycero-3-phosphocholine, a key substrate for the generation of the platelet-activating factor in the tissue.