Sequence Determinants for Regulated Degradation of Yeast 3-Hydroxy-3-Methylglutaryl-CoA Reductase, an Integral Endoplasmic Reticulum Membrane Protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27–54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.

Bidirectional, experience-dependent regulation of N-methyl-d-aspartate receptor subunit composition in the rat visual cortex during postnatal development

In the visual cortex, as elsewhere, N-methyl-d-aspartate receptors (NMDARs) play a critical role in triggering long-term, experience-dependent synaptic plasticity. Modifications of NMDAR subunit composition alter receptor function, and could have a large impact on the properties of synaptic plasticity. We have used immunoblot analysis to investigate the effects of age and visual experience on the expression of different NMDAR subunits in synaptoneurosomes prepared from rat visual cortices. NMDARs at birth are comprised of NR2B and NR1 subunits, and, over the first 5 postnatal weeks, there is a progressive inclusion of the NR2A subunit. Dark rearing from birth attenuates the developmental increase in NR2A. Levels of NR2A increase rapidly (in <2 hr) when dark-reared animals are exposed to light, and decrease gradually over the course of 3 to 4 days when animals are deprived of light. These data reveal that NMDAR subunit composition in the visual cortex is remarkably dynamic and bidirectionally regulated by sensory experience. We propose that NMDAR subunit regulation is a mechanism for experience-dependent modulation of synaptic plasticity in the visual cortex, and serves to maintain synaptic strength within an optimal dynamic range.

Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

Down-Regulation of Platelet Endothelial Cell Adhesion Molecule-1 Results in Thrombospondin-1 Expression and Concerted Regulation of Endothelial Cell Phenotype

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell–cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND.3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a “switch” that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.

Regulation of fatty acid homeostasis in cells: Novel role of leptin

It is proposed that an important function of leptin is to confine the storage of triglycerides (TG) to the adipocytes, while limiting TG storage in nonadipocytes, thus protecting them from lipotoxicity. The fact that TG content in nonadipocytes normally remains within a narrow range, while that of adipocytes varies enormously with food intake, is consistent with a system of TG homeostasis in normal nonadipocytes. The facts that when leptin receptors are dysfunctional, TG content in nonadipocytes such as islets can increase 100-fold, and that constitutively expressed ectopic hyperleptinemia depletes TG, suggest that leptin controls the homeostatic system for intracellular TG. The fact that the function and viability of nonadipocytes is compromised when their TG content rises above or falls below the normal range suggests that normal homeostasis of their intracellular TG is critical for optimal function and to prevent lipoapoptosis. Thus far, lipotoxic diabetes of fa/fa Zucker diabetic fatty rats is the only proven lipodegenerative disease, but the possibility of lipotoxic disease of skeletal and/or cardiac muscle may require investigation, as does the possible influence of the intracellular TG content on autoimmune and neoplastic processes.

Topology of allosteric regulation of lactose permease

Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog β-d-galactopyranosyl 1-thio-β-d-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.

CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression.

Cultured human umbilical vein endothelial cells (EC) constitutively express a low level of CD40 antigen as detected by monoclonal antibody binding and fluorescence flow cytometric quantitation. The level of expression on EC is increased about 3-fold following 24 h treatment with optimal concentrations of tumor necrosis factor, interleukin 1, interferon beta, or interferon gamma; both interferons show greater than additive induction of CD40 when combined with tumor necrosis factor or interleukin 1. Expression of CD40 increases within 8 h of cytokine treatment and continues to increase through 72 h. A trimeric form of recombinant murine CD40 ligand acts on human EC to increase expression of leukocyte adhesion molecules, including E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1. CD40 may be detected immunocytochemically on human microvascular EC in normal skin. We conclude that endothelial CD40 may play a role as a signaling receptor in the development of T-cell-mediated inflammatory reactions.

Regulation of T-cell antigen receptor (TCR) alpha-chain expression by TCR beta-chain transcripts.

The TCR is an alpha beta heterodimer, a part of the multimeric structure through which physiological T-cell activation occurs. The expression of TCR alpha chain is greatly diminished in a beta-chain-deficient mutant Jurkat cell line (J.RT3-T3.5). The relationship between the expression of the TCR alpha and beta chains has been examined by stable transfection of a series of TCR beta-chain mutant constructs into this mutant cell line. The level of alpha-chain transcript was dramatically upregulated by the expression of the beta chain and specifically by a transcript of the beta-chain variable region alone, including a transcript in which the ATG start codon was mutated. The downregulation of the endogenous alpha-chain transcripts in mutants cells lacking complete beta-chain transcripts occurred primarily at the posttranscriptional level. This evidence for a regulatory function of the TCR beta-chain gene represents an unusual regulatory pathway in which the transcript of one gene is required for the optimal expression of another gene.