The RGG domain of Npl3p recruits Sky1p through docking interactions

The SR protein kinase in yeast, Sky1p, phosphorylates yeast SR-like protein, Npl3p, at a single serine residue located at its C terminus. We report here the X-ray crystal structure of Sky1p bound to a substrate peptide and ADP. Surprisingly, an Npl3p-derived substrate peptide occupies a groove 20 A away from the kinase active site. In vitro studies support the substrate-docking role of this groove. Mutagenesis and binding studies reveal that multiple degenerate short peptide motifs located within the RGG domain of Npl3p serve as the substrate docking motifs. However, a single docking motif is sufficient for its stable interaction with the kinase. Methylation of the docking motifs abolishes kinase binding and phosphorylation of Npl3p. Remarkably, removal of the docking groove in the kinase or the docking motifs of the substrate does not reduce the overall catalytic efficiency of the phosphorylation reaction in any significant manner. We suggest that docking interaction between Sky1p and Npl3p is essential for substrate recruitment and binding specificity.     

The membrane anchor R7BP controls the proteolytic stability of the striatal specific RGS protein, RGS9-2

A member of the RGS (regulators of G protein signaling) family, RGS9-2 is a critical regulator of G protein signaling pathways that control locomotion and reward signaling in the brain. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with its newly discovered partner, R7BP (R7 family binding protein). Interaction with R7BP is important for the subcellular targeting of RGS9-2, which in native neurons is found in plasma membrane and its specializations, postsynaptic densities. Here we report that R7BP plays an additional important role in determining proteolytic stability of RGS9-2. We have found that co-expression with R7BP dramatically elevates the levels of RGS9-2 and its constitutive subunit, Gbeta5. Measurement of the RGS9-2 degradation kinetics in cells indicates that R7BP markedly reduces the rate of RGS9-2.Gbeta5 proteolysis. Lentivirus-mediated RNA interference knockdown of the R7BP expression in native striatal neurons results in the corresponding decrease in RGS9-2 protein levels. Analysis of the molecular determinants that mediate R7BP/RGS9-2 binding to result in proteolytic protection have identified that the binding site for R7BP in RGS proteins is formed by pairing of the DEP (Disheveled, EGL-10, Pleckstrin) domain with the R7H (R7 homology), a domain of previously unknown function that interacts with four putative alpha-helices of the R7BP core. These findings provide a mechanism for the regulation of the RGS9 protein stability in the striatal neurons.     

Preferential infection shortens the life span of human immunodeficiency virus-specific CD4+ T cells in vivo

CD4(+) T-cell help is essential for effective immune responses to viruses. In human immunodeficiency virus (HIV) infection, CD4(+) T cells specific for HIV are infected by the virus at higher frequencies than other memory CD4(+) T cells. Here, we demonstrate that HIV-specific CD4(+) T cells are barely detectable in most infected individuals and that the corresponding CD4(+) T cells exhibit an immature phenotype compared to both cytomegalovirus (CMV)-specific CD4(+) T cells and other memory CD4(+) T cells. However, in two individuals, we observed a rare and diametrically opposed pattern in which HIV-specific CD4(+) T-cell populations of large magnitude exhibited a terminally differentiated immunophenotype; these cells were not preferentially infected in vivo. Clonotypic analysis revealed that the HIV-specific CD4(+) T cells from these individuals were cross-reactive with CMV. Thus, preferential infection can be circumvented in the presence of cross-reactive CD4(+) T cells driven to maturity by coinfecting viral antigens, and this physical proximity rather than activation status per se is an important determinant of preferential infection based on antigen specificity. These data demonstrate that preferential infection reduces the life span of HIV-specific CD4(+) T cells in vivo and thereby compromises the generation of effective immune responses to the virus itself; further, this central feature in the pathophysiology of HIV infection can be influenced by the cross-reactivity of responding CD4(+) T cells.     

Placental insufficiency leads to developmental hypertension and mesenteric artery dysfunction in two generations of Sprague-Dawley rat offspring

It is generally accepted that preeclampsia results from reduction in perfusion to the uteroplacental unit leading to maternal hypertension and fetal growth restriction. Placental insufficiency creates an environment of fetal undernutriton, predisposing the fetus to the development of adult disease. In this study, we characterized the development and perpetuation of hypertension in two generations of male and female offspring subjected to an environment of fetal undernutrition via reduced uteroplacental perfusion pressure. Further, we examined vascular responses of resistance arteries in these animals to determine the influence of placental insufficiency on the development and perpetuation of hypertension. Experimental dams underwent a surgical procedure to reduce uteroplacental perfusion pressure, with resulting offspring comprising the first generation (F1). One male and one female from each of the F1 experimental litters served as breeders of the second generation (F2). Weekly systolic blood pressure measurements were obtained from 4 to 24 wk in control, F1, and F2 offspring. Vascular responsiveness to the vasoconstrictors phenylephrine and potassium chloride and the vasorelaxants acetylcholine and sodium nitroprusside was determined in the three offspring groups at 6, 9, and 12 wk of age. Our findings indicate that placental insufficiency during a critical developmental window in late gestation leads to hypertension in juvenile Sprague-Dawley rat offspring and is perpetuated in a second generation of offspring in a gender-specific manner. Further, exposure to placental insufficiency during late gestation leads to developmental alterations characterized by vascular hyperresponsiveness, perpetuated to a second generation of offspring in the absence of persistent environmental stimuli, contributing to hypertension.     

Tyrosine hydroxylase-synthesizing cells in the hypothalamus of prairie voles (Microtus ochrogaster): sex differences in the anteroventral periventricular preoptic area and effects of adult gonadectomy or neonatal gonadal hormones

The vertebrate hypothalamus and surrounding region contain a large population of cells expressing tyrosine hydroxylase (TH), the rate limiting enzyme for synthesis of dopamine and other catecholamines. Some of these populations are sexually dimorphic in rats. We here examined sex differences in TH-immunoreactive populations in the forebrain of gonadally intact and gonadectomized prairie voles (Microtus ochrogaster), a species that sometimes shows unusual sexual differentiation of brain and behavior. A sex difference was found in the anteroventral periventricular preoptic area (AVPV; likely analogous to the rat rostral A14) only in gonadectomized subjects, which was due to a 50% reduction in the number of TH-immunoreactive cells after castration in males. There was no significant sex difference or effects of gonadectomy on the number of TH-immunoreactive cells in the anteroventral preoptic area (AVP), periventricular anterior hypothalamus (caudal A14), arcuate nucleus (A12), zona incerta (A13), or posterodorsal hypothalamus (A11). In a second experiment, testosterone propionate (TP; 500 microg), diethylstilbestrol (DES; 1 microg), or estradiol benzoate (EB; 30 microg) injected daily during the first week after birth each significantly reduced later TH expression in the AVPV of females by approximately 40-65% compared to oil-treated controls. Unlike rats, therefore, a sex difference in TH expression in the prairie vole AVPV is found only after removal of circulating gonadal hormones in males. Furthermore, unlike our previous findings on the generation of sex differences in extra-hypothalamic arginine-vasopressin expression in prairie voles, TH expression in the AVPV of female prairie voles can be highly masculinized by neonatal exposure to either aromatizable androgens or estrogens.     

The products of the herpes simplex virus type 1 immediate-early US1/US1.5 genes downregulate levels of S-phase-specific cyclins and facilitate virus replication in S-phase Vero cells

Herpes simplex virus type 1 ICP22-/U(S)1.5- mutants initiate viral gene expression in all cells; however, in most cell types, the replication process stalls due to an inability to express gamma2 late proteins. Although the function of ICP22/U(S)1.5 has not been established, it has been suggested that these proteins activate, induce, or repress the activity of cellular proteins during infection. In this study, we hypothesized that cell cycle-associated proteins are targets of ICP22/U(S)1.5. For this purpose, we first isolated and characterized an ICP22-/U(S)1.5- mutant virus, 22/n199. Like other ICP22-/U(S)1.5- mutants, 22/n199 replicates in a cell-type-specific manner and fails to induce efficient gamma2 late gene expression in restrictive cells. Although synchronization of restrictive human embryonic lung cells in each phase of the cell cycle did not overcome the growth restrictions of 22/n199, synchronization of permissive Vero cells in S phase rendered them less able to support 22/n199 plaque formation and replication. Consistent with this finding, expression of cellular S-phase cyclins was altered in an ICP22/U(S)1.5-dependent manner specifically when S-phase Vero cells were infected. Collectively, these observations support the notion that ICP22/U(S)1.5 deregulates the cell cycle upon infection of S-phase permissive cells by altering expression of key cell cycle regulatory proteins either directly or indirectly.     

Homer 1 mediates store- and inositol 1,4,5-trisphosphate receptor-dependent translocation and retrieval of TRPC3 to the plasma membrane

Store-operated Ca(2+) channels (SOCs) mediate receptor-stimulated Ca(2+) influx. Accumulating evidence indicates that members of the transient receptor potential (TRP) channel family are components of SOCs in mammalian cells. Agonist stimulation activates SOCs and TRP channels directly and by inducing translocation of channels in intracellular vesicles to the plasma membrane (PM). The mechanism of TRP channel translocation in response to store depletion and agonist stimulation is not known. Here we use TRPC3 as a model to show that IP(3) and the scaffold Homer 1 (H1) regulate the rate of translocation and retrieval of TRPC3 from the PM. In resting cells, TRPC3 exists in TRPC3-H1b/c-IP(3)Rs complexes that are located in part at the PM and in part in intracellular vesicles. Binding of IP(3) to the IP(3)Rs dissociates the interaction between IP(3)Rs and H1 but not between H1 and TRPC3 to form IP(3)Rs-TRPC3-H1b/c. TIRFM and biotinylation assays show robust receptor- and store-dependent translocation of the TRPC3 to the PM and their retrieval upon termination of cell stimulation. The translocation requires depletion of stored Ca(2+) and is prevented by inhibition of the IP(3)Rs. In HEK293, dissociating the H1b/c-IP(3)R complex with H1a results in TRPC3 translocation to the PM, where it is spontaneously active. The TRPC3-H1b/c-IP(3)Rs complex is reconstituted by infusing H1c into these cells. Reconstitution is inhibited by IP(3). Deletion of H1 in mice markedly reduces the rates of translocation and retrieval of TRPC3. Conversely, infusion of H1c into H1(-/-) cells eliminates spontaneous channel activity and increases the rate of channel activation by agonist stimulation. The effects of H1c are inhibited by IP(3). These findings together with our earlier studies demonstrating gating of TRPC3 by IP(3)Rs were used to develop a model in which assembly of the TRPC3-H1b/c-IP(3)Rs complexes by H1b/c mediates both the translocation of TRPC3-containing vesicles to the PM and gating of TRPC3 by IP(3)Rs.     

Drosophila Plexin B is a Sema-2a receptor required for axon guidance

Plexin receptors play a crucial role in the transduction of axonal guidance events elicited by semaphorin proteins. In Drosophila, Plexin A (PlexA) is a receptor for the transmembrane semaphorin semaphorin-1a (Sema-1a) and is required for motor and central nervous system (CNS) axon guidance in the developing embryonic nervous system. However, it remains unknown how PlexB functions during neural development and which ligands serve to activate this receptor. Here, we show that plexB, like plexA, is robustly expressed in the developing CNS and is required for motor and CNS axon pathfinding. PlexB and PlexA serve both distinct and shared neuronal guidance functions. We observe a physical association between these two plexin receptors in vivo and find that they can utilize common downstream signaling mechanisms. PlexB does not directly bind to the cytosolic semaphorin signaling component MICAL (molecule that interacts with CasL), but requires MICAL for certain axonal guidance functions. Ligand binding and genetic analyses demonstrate that PlexB is a receptor for the secreted semaphorin Sema-2a, suggesting that secreted and transmembrane semaphorins in Drosophila use PlexB and PlexA, respectively, for axon pathfinding during neural development. These results establish roles for PlexB in central and peripheral axon pathfinding, define a functional ligand for PlexB, and implicate common signaling events in plexin-mediated axonal guidance.