B族G蛋白偶联受体PAC1的N端基序HSDCIF对其二聚化和上膜运输的影响

B族G蛋白偶联受体（G protein-coupled receptors, GPCRs）PAC1是垂体腺苷酸环化酶激活多肽（pituitary adenylate cyclase activating polypeptide, PACAP）的特异受体,介导PACAP神经保护等功能,是神经系统疾病药物开发的重要靶点之一. HSDCIF（His-Ser-Asp-Cys-Ile-Phe）为位于PAC1的N端胞外1区（extracellar domain 1, EC1）的一段短肽序列,与特定负责激活PAC1受体的激动域PACAP（1-6）具有极高的同源性.利用基因敲除技术构建出缺陷HSDCIF基序的PAC1突变体（简称D PAC1）|利用基因工程原理和技术构建系列真核表达重组载体,包括融合了增强型黄色荧光蛋白（enhanced yellow fluorescent protein, EYFP）的表达载体D-PAC1-EYFP|用于生物发光能量转移（bioluminescence resonance energy transfer, BRET）检测的D-PAC1-Rluc|以及用于双分子荧光互补（bimolecular fluorescence complementation, BiFC）实验的D-PAC1-EYFP/N和D-PAC1-EYFP/C.免疫荧光检测（immunofluorescence assay）测定D PAC1的表达|荧光共聚焦显微观察D-PAC1的细胞运输,然后通过 Western印迹、BRET与BiFC方法来检测D PAC1的二聚化情况,综合评价HSDCIF基序对PAC1二聚化和在细胞中定位的影响.检测结果显示,缺陷HSDCIF基序的突变体D PAC1不能发生二聚化,也不能正常的进行上膜运输,而是滞留在内质网中,同时外源化学合成的寡肽HSDCIF可以竞争性地抑制正常PAC1的二聚化.     

荧光互补与光能量共振转移检测B类G蛋白偶联受体PAC1二聚化

G蛋白偶联受体（G—protein couple receptors,GPCRs）是最大的超家族膜受体,其中它的B家族成员垂体腺苷酸环化酶激活肽（PACl）是垂体腺苷酸环化酶激动多肽（PAcAP）的特异受体,介导PAcAP神经保护等功能,是神经系统疾病药物开发的重要靶点之一。二聚化或寡聚化是GPCRs普遍存在的现象,但是目前尚没有关于PACl形成同源二聚体或寡聚体的报道。为了验证PACl也能进行同源二聚化,该文采用生物发光能量转移（bioluminescence resonance energy transfer,BRET）方法进行检测,结果显示不同浓度梯度共转染中国仓鼠卵巢细胞（Chinesehamsterovary,CHO）的PACl一Rluc与PACl一EYFP重组载体,在底物腔肠素h（coelenterazineh）作用下呈现明显的BRET信号。双分子荧光互补（BiFc）检测显示,带有EYFPN端基因标记的PACl与带有EYFPC端基因标记的PACl共转染CHO细胞,能呈现完整的EYFP荧光信号。同时,Westemblot检测也显示,高表达PACl的细胞中可检测到JPACl二聚体的大分子。因此,PACl是能够进行正常同源二聚化的,这个发现将为后续神经损伤药物的开发奠定全新的理论基础,同时也为其他GPCRs同源二聚化的研究起到启发和借鉴作用。     

Improved Methodical Approach for Quantitative BRET Analysis of G Protein Coupled Receptor Dimerization

G Protein Coupled Receptors (GPCR) can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET) measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V₂ vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β₂ adrenergic and CB₁ cannabinoid receptors.     

HIV-1 Nef Dimerization Is Required for Nef-Mediated Receptor Downregulation and Viral Replication

Nef, a human immunodeficiency virus type 1 (HIV-1) accessory factor capable of interaction with a diverse array of host cell signaling molecules, is essential for high-titer HIV replication and AIDS progression. Previous biochemical and structural studies have suggested that Nef may form homodimers and higher-order oligomers in HIV-infected cells, which may be required for both immune and viral receptor downregulation as well as viral replication. Using bimolecular fluorescence complementation, we provide the first direct evidence for Nef dimers within HIV host cells and identify the structural requirements for dimerization in vivo. Bimolecular fluorescence complementation analysis shows that the multiple hydrophobic and electrostatic interactions found within the dimerization interface of the Nef X-ray crystal structure are essential for dimerization in cells. Nef dimers localized to the plasma membrane as well as the trans-Golgi network, two subcellular localizations essential for Nef function. Mutations in the Nef dimerization interface dramatically reduced both Nef-induced CD4 downregulation and HIV replication. Viruses expressing dimerization-defective Nef mutants were disabled to the same extent as HIV that fails to express Nef in terms of replication. These results identify the Nef dimerization region as a potential molecular target for antiretroviral drug discovery.     

The N-Terminal GYPSY Motif Is Required for Pilin-Specific Sortase SrtC1 Functionality in Lactobacillus rhamnosus Strain GG

Predominantly identified in pathogenic Gram-positive bacteria, sortase-dependent pili are also found in commensal species, such as the probiotic-marketed strain Lactobacillus rhamnosus strain GG. Pili are typically associated with host colonization, immune signalling and biofilm formation. Comparative analysis of the N-terminal domains of pilin-specific sortases from various piliated Gram-positive bacteria identified a conserved motif, called GYPSY, within the signal sequence. We investigated the function and role of the GYPSY residues by directed mutagenesis in homologous (rod-shaped) and heterologous (coccoid-shaped) expression systems for pilus formation. Substitutions of some of the GYPSY residues, and more specifically the proline residue, were found to have a direct impact on the degree of piliation of Lb. rhamnosus GG. The present findings uncover a new signalling element involved in the functionality of pilin-specific sortases controlling the pilus biogenesis of Lb. rhamnosus GG and related piliated Gram-positive species.     

N-Terminal T4 Lysozyme Fusion Facilitates Crystallization of a G Protein Coupled Receptor

A highly crystallizable T4 lysozyme (T4L) was fused to the N-terminus of the β₂ adrenergic receptor (β₂AR), a G-protein coupled receptor (GPCR) for catecholamines. We demonstrate that the N-terminal fused T4L is sufficiently rigid relative to the receptor to facilitate crystallogenesis without thermostabilizing mutations or the use of a stabilizing antibody, G protein, or protein fused to the 3rd intracellular loop. This approach adds to the protein engineering strategies that enable crystallographic studies of GPCRs alone or in complex with a signaling partner.     

N-Terminal Domain of Borna Disease Virus G (p56) Protein Is Sufficient for Virus Receptor Recognition and Cell Entry

Borna disease virus (BDV) surface glycoprotein (GP) (p56) has a predicted molecular mass of 56 kDa. Due to extensive posttranslational glycosylation the protein migrates as a polypeptide of 84 kDa (gp84). The processing of gp84 by the cellular protease furin generates gp43, which corresponds to the C-terminal part of gp84. Both gp84 and gp43 have been implicated in viral entry involving receptor-mediated endocytosis and pH-dependent fusion. We have investigated the domains of BDV p56 involved in virus entry. For this, we used a pseudotype approach based on a recently developed recombinant vesicular stomatitis virus (VSV) in which the gene for green fluorescent protein was substituted for the VSV G protein gene (VSV Delta G*). Complementation of VSV Delta G* with BDV p56 resulted in infectious VSV Delta G* pseudotypes that contained both BDV gp84 and gp43. BDV-VSV chimeric GPs that contained the N-terminal 244 amino acids of BDV p56 and amino acids 421 to 511 of VSV G protein were efficiently incorporated into VSV Delta G* particles, and the resulting pseudotype virions were neutralized by BDV-specific antiserum. These findings indicate that the N-terminal part of BDV p56 is sufficient for receptor recognition and virus entry.     

Dimerization of Corticotropin-Releasing Factor Receptor Type 1 Is Not Coupled to Ligand Binding

As described previously, receptor dimerization of G protein-coupled receptors may influence signaling, trafficking, and regulation in vivo. Up to now, most studies aiming at the possible role of receptor dimerization in receptor activation and signal transduction are focused on class A GPCRs. In the present work, the dimerization behavior of the corticotropin-releasing factor receptor type 1 (CRF₁R), which belongs to class B of GPCRs and plays an important role in coordination of the immune response, stress, and learning behavior, was investigated by using fluorescence resonance energy transfer (FRET). For this purpose, we generated fusion proteins of CRF₁R tagged at their C-terminus to a cyan or yellow fluorescent protein, which can be used as a FRET pair. Binding studies verified that the receptor constructs were able to bind their natural ligands in a manner comparable with the wild-type receptor, whereas cAMP accumulation proved the functionality of the constructs. In microscopic studies, a dimerization of the CRF₁R was observed, but the addition of either CRF-related agonists or antagonists did not show any dose-related increase of the observed FRET signal, indicating that the dimer-monomer ratio is not changed on addition of ligand.     

Dimerization of Toll-like Receptor 3 (TLR3) Is Required for Ligand Binding

TLR3 (Toll-like receptor 3) recognizes dsRNA, a potent indicator of viral infection. The extracellular domain of TLR3 dimerizes when it binds dsRNA, and the crystal structure of the dimeric complex reveals three sites of interaction on each extracellular domain, two that bind dsRNA and one that is responsible for dimer formation. The goal of this study was to determine which amino acid residues are essential for forming a stable receptor·ligand complex and whether dimerization of TLR3 is required for dsRNA binding. Using a novel ELISA to analyze dsRNA binding by mutant TLR3 constructs, we identified the essential interacting residues and determined that the simultaneous interaction of all three sites is required for ligand binding. In addition, we show that TLR3 is unable to bind dsRNA when dimerization is prevented by mutating residues in the dimerization site or by immobilizing TLR3 at low density. We conclude that dimerization of TLR3 is essential for ligand binding and that the three TLR3 contact sites individually interact weakly with their binding partners but together form a high affinity receptor·ligand complex.     

The Arabidopsis Receptor Kinase ZAR1 Is Required for Zygote Asymmetric Division and Its Daughter Cell Fate

Asymmetric division of zygote is critical for pattern formation during early embryogenesis in plants and animals. It requires integration of the intrinsic and extrinsic cues prior to and/or after fertilization. How these cues are translated into developmental signals is poorly understood. Here through genetic screen for mutations affecting early embryogenesis, we identified an Arabidopsis mutant, zygotic arrest 1 (zar1), in which zygote asymmetric division and the cell fate of its daughter cells were impaired. ZAR1 encodes a member of the RLK/Pelle kinase family. We demonstrated that ZAR1 physically interacts with Calmodulin and the heterotrimeric G protein Gβ, and ZAR1 kinase is activated by their binding as well. ZAR1 is specifically expressed micropylarly in the embryo sac at eight-nucleate stage and then in central cell, egg cell and synergids in the mature embryo sac. After fertilization, ZAR1 is accumulated in zygote and endosperm. The disruption of ZAR1 and AGB1 results in short basal cell and an apical cell with basal cell fate. These data suggest that ZAR1 functions as a membrane integrator for extrinsic cues, Ca²⁺ signal and G protein signaling to regulate the division of zygote and the cell fate of its daughter cells in Arabidopsis.     

Myosin IIb-dependent Regulation of Actin Dynamics Is Required for N-Methyl-d-aspartate Receptor Trafficking during Synaptic Plasticity

N-Methyl-d-aspartate receptor (NMDAR) synaptic incorporation changes the number of NMDARs at synapses and is thus critical to various NMDAR-dependent brain functions. To date, the molecules involved in NMDAR trafficking and the underlying mechanisms are poorly understood. Here, we report that myosin IIb is an essential molecule in NMDAR synaptic incorporation during PKC- or θ burst stimulation-induced synaptic plasticity. Moreover, we demonstrate that myosin light chain kinase (MLCK)-dependent actin reorganization contributes to NMDAR trafficking. The findings from additional mutual occlusion experiments demonstrate that PKC and MLCK share a common signaling pathway in NMDAR-mediated synaptic regulation. Because myosin IIb is the primary substrate of MLCK and can regulate actin dynamics during synaptic plasticity, we propose that the MLCK- and myosin IIb-dependent regulation of actin dynamics is required for NMDAR trafficking during synaptic plasticity. This study provides important insights into a mechanical framework for understanding NMDAR trafficking associated with synaptic plasticity.     

A DNA Damage-Regulated BRCT-Containing Protein, TopBP1, Is Required for Cell Survival

BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.     

The BiP Cochaperone ERdj4 Is Required for B Cell Development and Function

ERdj4 is a BiP cochaperone regulated by the unfolded protein response to facilitate degradation of unfolded and/or misfolded proteins in the endoplasmic reticulum. As the unfolded protein response plays a critical role in B cell maturation and antibody production, ERdj4 gene trap mice were generated to determine if this chaperone was required for B cell homeostasis. Homozygosity for the trapped allele resulted in hypomorphic expression of ERdj4 in bone marrow cells and abnormal development of hematopoietic lineages in the bone marrow. The number of myeloid cells was increased, while the number of erythroid and B lymphoid cells was reduced in ERdj4 gene trap mice compared to controls. An intrinsic B cell defect was identified that decreased survival of B cell precursors including large and small pre-B, and immature B cells. Consistent with impaired B lymphopoiesis, the number of mature follicular B cells was reduced in both the bone marrow and spleen of ERdj4 gene trap mice. Paradoxically, unchallenged ERdj4 gene trap mice showed non-specific hypergammaglobulinemia and gene trap B cells exhibited increased proliferation, survival and isotype switching in response to LPS stimulation. Although ERdj4 gene trap mice responded normally to T cell-independent antigen, they failed to mount a specific antibody response to T cell-dependent antigen in vivo. Collectively, these findings demonstrate that the chaperone activity of ERdj4 is required for survival of B cell progenitors and normal antibody production.