Physiological roles of clathrin adaptor AP complexes: lessons from mutant animals

Clathrin-associated adaptor protein (AP) complexes play a key role in the transport of proteins, by regulating the formation of transport vesicles as well as cargo selection, between organelles of the post-Golgi network, namely, the trans-Golgi network (TGN), endosomes, lysosomes and the plasma membrane. Evidence has been accumulating for the physiological importance of AP complexes. Deficiency in AP-1A or AP-2 results in embryonic lethality in mice, indicating that these AP complexes are essential for normal development of embryos in mammals. In contrast, mutations in the genes encoding subunits of AP-3A cause an autosomal recessive disorder, Hermansky-Pudlak syndrome in human and its disease models in mice. Knockout mice for the neuron-specific AP-3B suffer from epileptic seizure. Further studies on the physiological and pathological aspects of AP complexes will not only be beneficial for better understanding of developmental biology and medical sciences, but also deepen our insight into the molecular mechanisms of vesicular traffic.     

The steroid and xenobiotic receptor negatively regulates B-1 cell development in the fetal liver

The steroid and xenobiotic receptor (SXR) (also known as pregnane X receptor or PXR) is a broad-specificity nuclear hormone receptor that is well known for its role in drug and xenobiotic metabolism. SXR is activated by a wide variety of endobiotics, dietary compounds, pharmaceuticals, and xenobiotic chemicals. SXR is expressed at its highest levels in the liver and intestine yet is found in lower levels in other tissues, where its roles are less understood. We previously demonstrated that SXR(-/-) mice demonstrate elevated nuclear factor (NF)-κB activity and overexpression of NF-κB target genes and that SXR(-/-) mice develop lymphoma derived from B-1 lymphocytes in an age-dependent manner. In this work, we show that fetal livers in SXR(-/-) mice display elevated expression of NF-κB target genes and possess a significantly larger percentage of B-1 progenitor cells in the fetal liver. Furthermore, in utero activation of SXR in wild-type mice reduces the B-1 progenitor populations in the embryonic liver and reduces the size of the B-1 cell compartment in adult animals that were treated in utero. This suggests that activation of SXR during development may permanently alter the immune system of animals exposed in utero, demonstrating a novel role for SXR in the generation of B-1 cell precursors in the fetal liver. These data support our previous findings that SXR functions as a tumor suppressor in B-1 lymphocytes and establish a unique role for SXR as a modulator of developmental hematopoiesis in the liver.     

Modulation of renal disease in MRL/lpr mice genetically deficient in the alternative complement pathway factor B

In systemic lupus erythematosus, the renal deposition of complement-containing immune complexes initiates an inflammatory cascade resulting in glomerulonephritis. Activation of the classical complement pathway with deposition of C3 is pathogenic in lupus nephritis. Although the alternative complement pathway is activated in lupus nephritis, its role in disease pathogenesis is unknown. To determine the role of the alternative pathway in lupus nephritis, complement factor B-deficient mice were backcrossed to MRL/lpr mice. MRL/lpr mice develop a spontaneous lupus-like disease characterized by immune complex glomerulonephritis. We derived complement factor B wild-type (B+/+), homozygous knockout (B-/-), and heterozygous (B+/-) MRL/lpr mice. Compared with B+/- or B+/+ mice, MRL/lpr B-/- mice developed significantly less proteinuria, less glomerular IgG deposition, and decreased renal scores as well as lower IgG3 cryoglobulin production and vasculitis. Serum C3 levels were normal in the B-/- mice compared with significantly decreased levels in the other two groups. These results suggest that: 1) factor B plays an important role in the pathogenesis of glomerulonephritis and vasculitis in MRL/lpr mice; and 2) activation of the alternative pathway, either by the amplification loop or by IgA immune complexes, has a prominent effect on serum C3 levels in this lupus model.     

Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus-Merzbacher disease

The mouse mutants mocha and pearl are deficient in the AP-3 delta and beta3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the beta3 and mu3 subunits coassemble into a heterodimer, whereas the sigma3 subunit remains monomeric. In pearl cells, the delta and sigma3 subunits coassemble into a heterodimer, whereas mu3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other. Pearl cell lines were generated that express beta3A, beta3B, a beta3Abeta2 chimera, two beta3A deletion mutants, and a beta3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The beta3Abeta2 chimera and the beta3A short deletion mutant gave partial functional rescue, whereas the beta3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.     

Immunoglobulin light chains dictate vesicular transport-dependent and -independent routes for IgM degradation by the ubiquitin-proteasome pathway

Degradation of IgM mu heavy chains in light chain-negative pre-B cells is independent of vesicular transport, as is evident by its insensitivity to brefeldin A or cell permeabilization. Conversely, by the same criteria, degradation of the secretory mu heavy chain in light chain-expressing B cells depends on vesicular transport. To investigate whether the presence of conventional light chains or the developmental stage of the B-lymphocytes dictates the degradative route taken by mu, we express in 70Z/3 pre-B cells either lambda ectopically or kappa by lipopolysaccharides-stimulated differentiation into B cells and show their assembly with mu heavy chains. The resulting sensitivity of mu degradation to brefeldin A and cell permeabilization demonstrates that conventional light chains, a hallmark of B cell differentiation, are necessary and sufficient to divert mu from a vesicular transport-independent to a vesicular transport-dependent degradative route. Although both routes converge at the ubiquitin-proteasome degradation pathway, only in light chain-expressing cells is vesicular transport a prerequisite for mu ubiquitination.     

The age-related failure of adaptive responses to contractile activity in skeletal muscle is mimicked in young mice by deletion of Cu,Zn superoxide dismutase

In muscle, aging is associated with a failure of adaptive responses to contractile activity, and this is hypothesized to play an important role in age-related loss of muscle mass and function. Mice lacking the Cu,Zn superoxide dismutase (Cu,ZnSOD, SOD1) show an accelerated, age-related loss of muscle mass and function. This work determined whether adult mice lacking Cu,ZnSOD (Sod1(-/-) mice) show a premature failure of adaptive responses to contractions in a similar manner to old wild-type (WT) mice. Adult Sod1(-/-) mice (6-8 months of age) had a ～30% reduction in gastrocnemius muscle mass compared with age-matched WT mice. This lower muscle mass was associated with an activation of DNA binding by NFκB and AP-1 at rest. Measurements of the activity of reactive oxygen species (ROS) in single fibres from the muscles of Sod1(-/-) mice at rest indicated an elevation in activity compared with fibres from WT mice. Following 15 min of isometric contractions, muscle fibres from WT mice showed an increase in the intracellular ROS activities and activation of NFκB and AP-1, but no changes in either ROS activity or NFκB and AP-1 activation were seen in the muscles of Sod1(-/-) mice following contractions. This pattern of changes mimics that seen in the muscles of old WT mice, suggesting that the attenuated responses to contractile activity seen in old mice result from chronic exposure to increased oxidant activity. Data support the use of the Sod1(-/-) mouse model to evaluate potential mechanisms that contribute to the loss of muscle mass and function in the elderly.     

Tagetone modulates the coupling of flunitrazepam and GABA binding sites at GABAA receptor from chick brain membranes

The effects of tagetone on flunitrazepam (FNTZ) binding to synaptosomal membranes from chick brains in the presence and absence of allosteric modulations induced by gamma-aminobutyric acid (GABA) were investigated. Tagetone, at 50 mu g/ml (final concentration), decreased the binding affinity of [3H]FNTZ to synaptosomal membranes form chick brain (Kd=3.34 +/- 0.36 nM without tagetone and Kd,t=5.86 +/- 0.86 nM with tagetone; p<0.05, two tailed Student's t-test) without affecting maximal binding (Bmax=488 +/- 24 fmoles/mg protein, and Bmax,t=500 +/- 25 fmoles/ mg protein in the absence and in the presence of tagetone respectively). The potency of GABA to stimulate [3H]FNTZ binding increased in the presence of tagetone (EC50 values were 2.78 and 1.12 mu M with and without tagetone respectively). GABA was able to decrease merocyanine Delta A570-610 values in a concentration dependent manner; half maximal effect was attained at a GABA concentration of 34 +/- 13 mu M. Tagetone, at a concentration of 50 mu g/ml and in the presence of GABA 30 mu M or 60 mu M, enhanced the ability of GABA alone on decreasing Delta A570-610. Tagetone alone did not change Delta A570-610 values. FNTZ, a well known GABA modulator, could also potentiate the effect of GABA. Theoretical calculations indicate that the effects on merocyanine Delta A value are mainly exerted at the membrane potential level (Delta Psim). The present results strongly suggest that tagetone affected the function of GABAA receptor in a complex way: on the one hand it impaired FNTZ binding; on the other hand tagetone improved both the coupling between FNTZ and GABA binding sites and it enhanced GABA-induced chloride permeability. Changes in the geometrical and electrostatic properties of the self-organized membrane structure may account for these effects of tagetone.     

Dual interaction of synaptotagmin with mu2- and alpha-adaptin facilitates clathrin-coated pit nucleation

The synaptic vesicle protein synaptotagmin was proposed to act as a major docking site for the recruitment of clathrin coats implicated in endocytosis, including the recycling of synaptic vesicles. We show here that the C2B domain of synaptotagmin binds mu2- and alpha-adaptin, two of the four subunits of the endocytic adaptor complex AP-2. mu2 represents the major interacting subunit of AP-2 within this complex. Its binding to synaptotagmin is mediated by a site in subdomain B that is distinct from the binding site for tyrosine-based sorting motifs located in subdomain A. The presence of the C2B domain of synaptotagmin at the surface of liposomes enhances the recruitment of AP-2 and clathrin. Conversely, perturbation of the interaction between synaptotagmin and AP-2 by synprint, the cytoplasmic synaptotagmin-binding domain of N-type calcium channels, inhibits transferrin internalization in living cells. We conclude that a dual interaction of synaptotagmin with the clathrin adaptor AP-2 plays a key physiological role in the nucleation of endocytic clathrin-coated pits.     

B-1 cell lymphoma in mice lacking the steroid and xenobiotic receptor, SXR

The steroid and xenobiotic receptor (SXR) is a broad-specificity nuclear hormone receptor that is highly expressed in the liver and intestine, where its primary function is to regulate drug and xenobiotic metabolism. SXR is expressed at lower levels in other tissues, where little is known about its physiological functions. We previously linked SXR with immunity and inflammation by showing that SXR antagonizes the activity of nuclear factor (NF)-κB in vitro and in vivo. SXR(-/-) mice demonstrate aberrantly high NF-κB activity and overexpression of NF-κB target genes. Here we show that SXR(-/-) mice develop B cell lymphoma in an age-dependent manner. SXR(-/-) mice develop multiple hyperplastic lymphoid foci composed of B-1a cells in the intestine, spleen, lymph nodes, peritoneal cavity, and blood. In all circumstances, these lymphocytes possess cell surface and molecular characteristics of either chronic lymphocytic leukemia or non-Hodgkin's lymphoma originating from B-1 lymphocytes. These results demonstrate a novel and unsuspected role for SXR signaling in the B-1 cell compartment, establish SXR as a tumor suppressor in B-1 cells, and may provide a link between metabolism of xenobiotic compounds and lymphomagenesis.     

GABA(B) receptors in 5-HT transporter- and 5-HT1A receptor-knock-out mice: further evidence of a transduction pathway shared with 5-HT1A receptors

The functional properties of GABA(B) receptors were examined in the dorsal raphe nucleus (DRN) and the hippocampus of knock-out mice devoid of the 5-HT transporter (5-HTT-/-) or the 5-HT(1A) receptor (5-HT(1A)-/-). Electrophysiological recordings in brain slices showed that the GABA(B) receptor agonist baclofen caused a lower hyperpolarization and neuronal firing inhibition of DRN 5-HT cells in 5-HTT-/- versus 5-HTT+/+ mice. In addition, [(35)S]GTP-gamma-S binding induced by GABA(B) receptor stimulation in the DRN was approximately 40% less in these mutants compared with wild-type mice. In contrast, GABA(B) receptors appeared functionally intact in the hippocampus of 5-HTT-/-, and in both this area and the DRN of 5-HT(1A)-knock-out mice. The unique functional changes of DRN GABA(B) receptors closely resembled those of 5-HT(1A) autoreceptors in 5-HTT-/- mice, further supporting the idea that both receptor types are coupled to a common pool of G-proteins in serotoninergic neurons.     

Basal activation of p70S6K results in adipose-specific insulin resistance in protein-tyrosine phosphatase 1B -/- mice

Although protein-tyrosine phosphatase 1B (PTP-1B) is a negative regulator of insulin action, adipose tissue from PTP-1B-/- mice does not show enhanced insulin-stimulated insulin receptor phosphorylation. Investigation of glucose uptake in isolated adipocytes revealed that the adipocytes from PTP-1B-/- mice have a significantly attenuated insulin response as compared with PTP-1B+/+ adipocytes. This insulin resistance manifests in PTP-1B-/- animals older than 16 weeks of age and could be partially rescued by adenoviral expression of PTP-1B in null adipocytes. Examination of adipose signaling pathways found that the basal p70S6K activity was at least 50% higher in adipose from PTP-1B-/- mice compared with wild type animals. The increased basal activity of p70S6K in PTP-1B-/- adipose correlated with decreases in IR substrate-1 protein levels and insulin-stimulated Akt/protein kinase B activity, explaining the decrease in insulin sensitivity even as insulin receptor phosphorylation was unaffected. The insulin resistance of the of the PTP-1B-/- adipocytes could also be rescued by treatment with rapamycin, suggesting that in adipose the loss of PTP-1B results in basal activation of mTOR (mammalian target of rapamycin) complex 1 leading to a tissue-specific insulin resistance.     

Signal-binding specificity of the mu4 subunit of the adaptor protein complex AP-4

The medium (mu) chains of the adaptor protein (AP) complexes AP-1, AP-2, and AP-3 recognize distinct subsets of tyrosine-based (YXXphi) sorting signals found within the cytoplasmic domains of integral membrane proteins. Here, we describe the signal-binding specificity and affinity of the medium subunit mu4 of the recently described adaptor protein complex AP-4. To elucidate the determinants of specificity, we screened a two-hybrid combinatorial peptide library using mu4 as a selector protein. Statistical analyses of the results revealed that mu4 prefers aspartic acid at position Y+1, proline or arginine at Y+2, and phenylalanine at Y-1 and Y+3 (phi). In addition, we examined the interaction of mu4 with naturally occurring YXXphi signals by both two-hybrid and in vitro binding analyses. These experiments showed that mu4 recognized the tyrosine signal from the human lysosomal protein LAMP-2, HTGYEQF. Using surface plasmon resonance measurements, we determined the apparent dissociation constant for the mu4-YXXphi interaction to be in the micromolar range. To gain insight into a possible role of AP-4 in intracellular trafficking, we constructed a Tac chimera bearing a mu4-specific YXXphi signal. This chimera was targeted to the endosomal-lysosomal system without being internalized from the plasma membrane.     

The AP-1A and AP-1B clathrin adaptor complexes define biochemically and functionally distinct membrane domains

Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD mu1A or mu1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B-containing membranes that were both distinct from AP-1A-positive TGN elements and more closely apposed to transferrin receptor-positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.     

T淋巴细胞在小鼠肾缺血再灌注损伤中的作用

目的研究T淋巴细胞在肾缺血再灌注损伤(IRI)导致的急性肾损害中的作用。方法BALB/c小鼠和BALB/c裸小鼠各24只,分别随机分为A1-4组和B1-4组,每组6只。双肾蒂阻断45 min后恢复血流建立肾IRI模型,假手术对照组I、RI后24、48和72 h时检测Scr、尿蛋白定量及肾病理学,A组检测脾T细胞亚群;对比BALB/c小鼠和BALB/c裸小鼠的肾功能下降、组织学损害程度以及脾T淋巴细胞亚群变化。结果A2-4组和B2-4组均有Scr和尿蛋白定量明显升高(P<0.05),且A组损害程度明显重于B组(P<0.05);A2-4组出现典型的IRI组织损害表现(P<0.05),B2-4组无明显IRI组织损害(P>0.05);A2-3组脾CD3 T细胞百分比较A1组升高(P<0.05),而CD4 /CD8 比值无明显变化(P>0.05)。结论T淋巴细胞是小鼠肾IRI导致急性肾损害的重要病理生理学因素。     

Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells

Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.     

Increased numbers of B-1 cells and enhanced responses against TI-2 antigen in mice lacking APS, an adaptor molecule containing PH and SH2 domains

APS (adaptor molecule containing PH and SH2 domains) is an intracellular adaptor protein that forms an adaptor family along with Lnk and SH2-B. While experiments using cultured cell lines have demonstrated that APS is phosphorylated in response to various stimuli, its in vivo functions remain unclear. We attempted to determine the physiological roles of APS by generating APS-deficient (APS(-/-)) mice. APS(-/-) mice were viable and fertile and showed no abnormalities or growth retardation. Immunologically, APS(-/-) mice showed normal development and distribution of lymphocytes and myeloid cells, except for increased numbers of B-1 cells in the peritoneal cavity. APS(-/-) mice exhibited an enhanced humoral immune response against trinitrophenol-Ficoll, a thymus-independent type 2 antigen, while APS(-/-) B-2 cells exhibited normal proliferative responses and tyrosine phosphorylation of intracellular proteins upon B-cell receptor (BCR) cross-linking. APS colocalized with filamentous actin (F-actin) accumulated during the capping of BCRs in APS-transgenic B cells. After BCR stimulation, F-actin contents were lower in APS(-/-) B-1 cells than in wild-type B-1 cells. Our results indicate that APS might have a novel regulatory role in actin reorganization and control of B-1 cell compartment size.     

B cell developmental requirement for the G alpha i2 gene

Null mutation of the Galphai2 trimeric G protein results in a discrete and profound mucosal disorder, including inflammatory bowel disease (IBD), attenuation of IL-10 expression, and immune function polarized to Th1 activity. Genetic and adoptive transfer experiments have established a role for B cells and IL-10 in mucosal immunologic homeostasis and IBD resistance. In this study, we addressed the hypothesis that Galphai2 is required for the development of IL-10-producing B cells. Galphai2(-/-) mice were reduced in the relative abundance of marginal zone (MZ), transitional type 2 (T2), and B-1a B cells and significantly increased in follicular mature and B-1b B cells. Reconstitution of RAG2(-/-) mice with Galphai2(-/-) bone marrow induced an IBD-like colitis and a deficiency in absolute numbers of MZ, T2, and B-1 B cells. Thus, the Galphai2(-/-) genotype in colitis susceptibility and B cell development involved a cis effect within the hemopoietic compartment. In vitro, the B cell population of Galphai2(-/-) mice was functionally deficient in LPS-induced proliferation and IL-10 production, consistent with the exclusive capacity of T2 and MZ cell subpopulations for LPS responsiveness. In vivo, Galphai2(-/-) mice were selectively impaired for the IgM response to T-independent type II, consistent with the relative depletion of MZ and peritoneal B-1 subpopulations. Collectively, these results reveal a selective role for Galphai2 in MZ and B-1 B cell development. Disorders of this Galphai2-dependent process in B cell development may represent a mechanism for IBD susceptibility.