钙敏感受体在淤胆型肝炎小鼠肝脏组织中的表达

[目的]探讨钙敏感受体（Calcium-sensingreceptor,CaR）在淤胆型肝炎小鼠肝组织中的表达。[方法]采用ANIT（80mg／kg,po）制备小鼠淤胆型肝炎模型,检测给予ANIT24h、48h后小鼠血清ALT、ALP及BA水平,HE染色观察肝脏病理组织学改变,免疫组化检测CaR在淤胆型肝炎小鼠肝组织中的表达。[结果]给予ANIT24h、48h后,小鼠血清ALT、ALP及BA水平逐渐升高,48h后明显上升;病理结果显示48h后,肝细胞坏死明显;免疫组化结果显示淤胆型肝炎小鼠肝组织中CaR的表达明显增强。[结论]CaR在淤胆型肝炎小鼠肝脏组织中存在高表达,且可能与淤胆的发生有关。     

Interdomain movements in metabotropic glutamate receptor activation

Many cell surface receptors are multimeric proteins, composed of several structural domains, some involved in ligand recognition, whereas others are responsible for signal transduction. In most cases, the mechanism of how ligand interaction in the extracellular domains leads to the activation of effector domains remains largely unknown. Here we examined how the extracellular ligand binding to the venus flytrap (VFT) domains of the dimeric metabotropic glutamate receptors activate the seven transmembrane (7TM) domains responsible for G protein activation. These two domains are interconnected by a cysteine-rich domain (CRD). We show that any of the four disulfide bridges of the CRD are required for the allosteric coupling between the VFT and the 7TM domains. More importantly, we show that a specific association of the two CRDs corresponds to the active state of the receptor. Indeed, a specific crosslinking of the CRDs with intersubunit disulfide bridges leads to fully constitutively active receptors, no longer activated by agonists nor by allosteric modulators. These data demonstrate that intersubunit movement at the level of the CRDs represents a key step in metabotropic glutamate receptor activation.     

Significance of calcium-sensing receptor expression in gastric cancer

Objective. The calcium-sensing receptor (CaSR) is known to have differential expression in various carcinomas and normal tissues. It has been shown to be involved in carcinogenesis or tumor suppression. However, its role in gastric cancer remains unknown. This study was performed to determine the CaSR expression level in gastric cancer and non-tumor gastric tissues and to examine the related clinicopathological factors. Materials and methods. Thirty-one pairs of gastric cancer tissues and matched non-tumor gastric tissues were obtained from surgical tissues after gastrectomy. Using real-time polymerase chain reaction, we measured CaSR mRNA expression. We evaluated the association between CaSR mRNA expression and clinicopathological variables based on the downregulation or upregulation of CaSR mRNA expression in gastric cancer tissues compared to those of matched non-tumor gastric tissues. By immunohistochemistry, we confirmed CaSR expression levels in gastric cancer tissues. Results. Downregulation of CaSR mRNA was observed in 77.4% of gastric cancer tissues compared to their matched normal tissues. Downregulated CaSR was associated with a tendency for deeper invasion into the proper muscle (p = 0.028) and more advanced stage (II–IV; p = 0.012). Conclusion. We conclude that downregulation of CaSR may contribute to the prevention or suppression of tumor outgrowth.     

钙敏感受体及其配体在血管系统中的作用和机制的研究进展

近年来多项研究发现，钙敏感受体（CaSR）在血管系统中表达并发挥重要作用。本文就CaSR的结构、功能、配体的分类及其在血管系统中作用作一简要综述。     

Extracellular calcium-sensing receptor distribution in osmoregulatory and endocrine tissues of the tilapia

The extracellular calcium-sensing receptor (CaSR) serves an important detector function in vertebrate Ca²⁺ homeostasis. In this study, we surveyed using immunohistochemistry the tissue and cellular distribution of the CaSR protein in the Mozambique tilapia (Oreochromis mossambicus) and the Japanese eel (Anguilla japonica). Specifically, we examined receptor expression in ion-transporting barrier tissues that may be directly responsive to extracellular Ca²⁺ levels, and in tissues that are implicated in endocrine signaling to homeostatic effectors such as Ca²⁺-transporting epithelia. In tilapia osmoregulatory tissues, CaSR protein is strongly expressed in proximal segments of renal tubule, but not in distal segments (where Na⁺,K⁺-ATPase is prominently expressed) or in glomeruli. The receptor was also localized in the ion-transporting mitochondria-rich cells of gill and in ion- and nutrient-transporting epithelia of middle and posterior intestine. Consistent with our earlier RT-PCR assessment of mRNA expression in tilapia, CaSR protein expression was salinity dependent in some osmoregulatory tissues. In tilapia pituitary gland, CaSR expression was observed in the rostral pars distalis (containing prolactin-secreting cells, and in the pars intermedia (containing somatolactin-secreting and melanocyte-stimulating hormone-secreting cells), with notably greater expression in the latter. In the eel, weak immunostaining was seen in the stanniocalcin-secreting cells of the corpuscles of Stannius. Olfactory lobe CaSR expression suggests an environment-sensing role for the receptor. Altogether, these findings support the involvement of CaSR in piscine Ca²⁺ homeostasis at the levels of environmental sensing, of integrative endocrine signaling through both hypercalcemic (prolactin, and perhaps somatolactin) and hypocalcemic (stanniocalcin) hormones, and of direct local regulation of Ca²⁺-transporting tissues.     

北京地区汉族青年女性钙敏感受体基因多态性分布及其与骨密度相关性的研究

目的了解北京地区汉族青年女性钙敏感受体（Calcium-sensing ReceptOF,CaSR）基因多态性的分布,探索其与汉族妇女峰值骨密度的相关性。方法分别采用突变分离PCR和错配PCR-RFLP方法检测北京地区202名无亲缘关系的汉族青年健康女性（20～35岁）受试者CaSR基因A986S、G990R基因型,测定其血钙（Ca）、磷（P）及甲状旁腺素（PTH）水平,以及肝肾功能等指标,应用双能X线骨密度仪（DXA）测定腰椎、股骨上段部位的骨密度（BMD）。结果（1）北京地区汉族女性中存在CaSR基因A986S、G990R多态性,AA、AS基因型频率分别为95．0％和5．0％,等位基因A和s的频率分别为97．5％和2．5％;RR、GR及GG基因型频率分别为21．3％、51．0％和27．7％,等位基因R和G的频率分别为46．8％和53．2％。（2）校正年龄、身高、体重、BMI及血Ca、PTH水平后,多因素协方差分析显示AA、AS基因型组之间L2-4、股骨颈、ward＇s三角及大转子部位的BMD无显著差异（P=0J090～0．671）;GG、GR及RR基因型组之间L2。、股骨颈、ward＇s三角及大转子部位的BMD也无显著差异（P=0．089～0．493）。结论（1）北京地区汉族青年女性中CaSR基因A986S、G990R多态性的分布频率,以A、G等位基因频率较高。（2）未发现CaSR基因A986S、G990R多态性与北京地区汉族青年女性峰值骨密度相关。     

Hypercalcaemic and hypocalcaemic conditions due to calcium-sensing receptor mutations

The extracellular calcium (Ca2+o)-sensing receptor (CaSR) enables the parathyroid glands and other CaSR-expressing cells involved in calcium homeostasis, such as the kidney and bone, to sense alterations in the level of Ca2+o and to respond with changes in function that are directed at normalizing the blood calcium concentration. Several disorders of Ca2+o sensing arise from inherited or acquired abnormalities that 'reset' the serum calcium concentration upwards or downwards. Heterozygous inactivating mutations of the CaSR produce a benign form of hypercalcaemia, termed 'familial hypocalciuric hypercalcaemia', while homozygous mutations produce a much more severe hypercalcaemic disorder resulting from marked hyperparathyroidism, called 'neonatal severe hyperparathyroidism'. Activating mutations cause a hypocalcaemic syndrome of varying severity, termed 'autosomal-dominant hypocalcaemia or hypoparathyroidism' as well as Bartter's syndrome type V. Calcimimetic CaSR activators and calcilytic CaSR antagonists have also been developed with potential for use in the treatment of these disorders.     

动脉粥样硬化对大鼠心肌钙敏感受体表达和细胞凋亡的影响

目的 观察高脂血症和动脉粥样硬化对大鼠心肌钙敏感受体(CaSR)表达和细胞凋亡的影响.方法 采用腹腔注射维生素(Vit)D3(6×105U/kg)+高脂饮食6周的方法,建立大鼠高脂血症和动脉粥样硬化模型.Wistar大鼠随机分为正常对照组(n=12)和动脉粥样硬化组(n=12).采用RT-PCR和Western blot分别观察CaSR、Bax、Bcl-2、easpase-3的mRNA和蛋白表达.TUNEL染色观察心肌细胞凋亡情况.光镜观察腹主动脉和心肌形态学变化.电镜观察心脏超微结构变化.紫外分光法检测乳酸脱氢酶、肌酸激酶、超氧化物岐化酶的活性和丙二醛的含量,电化学免疫发光法检测肌钙蛋白水平.结果 动脉粥样硬化组乳酸脱氢酶和肌酸激酶活性、丙二醛含量和肌钙蛋白水平、细胞凋亡指数以及CaSR、Bax和caspase-3的表达均高于对照组,而超氧化物岐化酶活性则低于对照组,Bcl-2表达低于对照组,心肌细胞超微结构损伤严重.结论 高脂血症和动脉粥样硬化可引起大鼠心肌CaSR的表达增加和细胞凋亡,其机制与心肌缺血所致的氧化应激有关.     

Normalization of serum calcium by cinacalcet in a patient with hypercalcaemia due to a de novo inactivating mutation of the calcium-sensing receptor

Familial benign hypocalciuric hypercalcaemia (FHH) results from a heterozygous inactivating mutation of the calcium-sensing receptor (CaR) and is characterized by hypercalcaemia, hypocalciuria and inappropriately normal plasma levels of parathyroid hormone. In a minority of patients, a loss of function mutation of the CaR results in severe hypercalcaemia associated with complications for which no effective surgical or medical treatment is available. We investigated the effects of the calcimimetic agent cinacalcet, an allosteric modulator of the CaR, in a 26-year-old man presenting with hypercalcaemia due to a de novo inactivating mutation of the CaR. Complicating features were recurrent psychosis and progressive severe osteoporosis. A single dose of either 30 or 60 mg of cinacalcet resulted in a 63-88% decline in plasma parathyroid hormone levels within 2 h of administration of the agent, reverting to baseline levels after 12 h. Normalization of serum calcium was more gradual but sustained for up to 12 months of treatment with a maintenance twice-daily oral dose of 60+30 mg cinacalcet. In addition to its beneficial effects in primary and secondary hyperparathyroidism, cinacalcet may open new therapeutic avenues in the management of a subset of patients with severe hypercalcaemia due to inactivating mutations of the CaR.     

Structural basis for glucose-6-phosphate activation of glycogen synthase

Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme’s response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.     

Regulation of human EGF receptor by lipids

The human epidermal growth factor receptor (EGFR) is a key representative of tyrosine kinase receptors, ubiquitous actors in cell signaling, proliferation, differentiation, and migration. Although the receptor is well-studied, a central issue remains: How does the compositional diversity and functional diversity of the surrounding membrane modulate receptor function? Reconstituting human EGFR into proteoliposomes of well-defined and controlled lipid compositions represents a minimal synthetic approach to systematically address this question. We show that lipid composition has little effect on ligand-binding properties of the EGFR but rather exerts a profound regulatory effect on kinase domain activation. Here, the ganglioside GM3 but not other related lipids strongly inhibited the autophosphorylation of the EGFR kinase domain. This inhibitory action of GM3 was only seen in liposomes compositionally poised to phase separate into coexisting liquid domains. The inhibition by GM3 was released by either removing the neuraminic acid of the GM3 headgroup or by mutating a membrane proximal lysine of EGFR (K642G). Our results demonstrate that GM3 exhibits the potential to regulate the allosteric structural transition from inactive to a signaling EGFR dimer, by preventing the autophosphorylation of the intracellular kinase domain in response to ligand binding.     

Functional interactions between the extracellular domain and the seven-transmembrane domain in Ca2+ receptor activation

We studied the activity of mutants involving the aminoterminal extracellular, seven-transmembrane (7TM) and carboxy-terminal tail domains of the human Ca²⁺ receptor to gain insight into the functional interactions between these domains during receptor activation. Missense mutations of highly conserved residues, D190 and E297, in the extracellular domain (ECD), and a mutation within part of the proximal carboxy-terminal tail, A877-880E, resulted in receptors with severely reduced response to Ca²⁺ despite adequate cell surface expression. Coexpression of either D190A or E297K mutants with A877-880E led to significant reconstitution of function. No such reconstitution occurred when D190A or E297K mutants were coexpressed with a truncation mutant possessing an intact amino-terminal extracellular and first transmembrane domain, despite evidence for heterodimerization and cell surface expression of the respective mutant receptors. In addition, no reconstitution of function was observed when D190A was coexpressed with a deletion Ca²⁺ receptor mutant lacking only a cysteine-rich region located in the ECD of the Ca²⁺ receptor (Ca-//-Ca). Moreover, coexpression of this Ca-//-Ca with A877-880E did not recover function. The results show that Ca2⁺ receptor extracellular and 7TM domains are discrete entities that can communicate within the context of a heterodimer composed of complementary mutant receptors. Two intact 7TM domains and two intact cysteine-rich regions appear to be required for such communication to occur. The results are discussed in the context of a speculative model of receptor structure and function. O. M. Hauache was supported by a grant (#10848-2) from FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil).     

Clues to understanding cold sensation: thermodynamics and electrophysiological analysis of the cold receptor TRPM8

The cold and menthol receptor, TRPM8, also designated CMR1, is a member of the transient receptor potential (TRP) family of excitatory ion channels. TRPM8 is a channel activated by cold temperatures, voltage, and menthol. In this study, we characterize the cold- and voltage-induced activation of TRPM8 channel in an attempt to identify the temperature- and voltage-dependent components involved in channel activation. Under equilibrium conditions, decreasing temperature has two effects. (i) It shifts the normalized conductance vs. voltage curves toward the left, along the voltage axis. This effect indicates that the degree of order is higher when the channel is in the open configuration. (ii) It increases the maximum channel open probability, suggesting that temperature affects both voltage-dependent and -independent pathways. In the temperature range between 18 degrees C and 25 degrees C, large changes in enthalpy (DeltaH=-112 kcal/mol) and entropy (DeltaS=-384 cal/mol K) accompany the activation process. The Q10 calculated in the same temperature range is 24. This thermodynamic analysis strongly suggests that the process of opening involves large conformational changes of the channel-forming protein. Therefore, the highly temperature-dependent transition between open and closed configurations is possible because enthalpy and entropy are both large and compensate each other. Our data also demonstrate that temperature and voltage interact allosterically to enhance channel opening.     

原发性甲状旁腺功能亢进症患者钙敏感受体基因多态性的分布及其与血钙水平的相关性

目的 研究原发性甲状旁腺功能亢进症患者中钙敏感受体（CASR）基因多态性的分布，及其与临床特征的相关性。方法 分别采用突变分离PCR（MS—PCR）和错配PCR—RFLP方法检测北京地区202名无亲缘关系的汉族健康妇女及45例原发性甲状旁腺功能亢进症（PHPT）患者CASR基因A986S、G990R基因型，测定血钙、游离钙、磷、甲状旁腺素（PTH）水平。结果 （1）北京地区年轻正常汉族妇女中存在CASR基因A986S、G990R多态性，A986S等位基因A和S的频率分别为0．975和0．025，G990R等位基因R和G的频率分别为0．468和0．532，符合Hardy—Weinberg平衡。（2）PHPr患者：①A986S等位基因A和S的频率分别为0．944和0．056，G990R等位基因R和G的频率分别为0．667和0．333[与健康妇女组比较G990R基因型分布和等位基因频率差异有统计学意义（P〈0．01）]。②AA、AS基因型组的血钙分别为（3．03±0．50）、（2．84±0．12）mmoL／L（P〈0．05），血游离钙分别为（1．56±0．32）、（1．42±0．07）mmol/L（P〈0．05），血磷分别为（0．71±0．18）、（0．69±0．07）mmo/L（P〉0．05），血PTH升高倍数分别为（13．13±11．26）、（9．25±7．78）倍（P〈0．05）。（3）PHPT患者GG＋GR与RR基因型组的血钙分别为（3．00±0．46）、（3．04±0．52）mmoL／L（P〈0．05），血游离钙分别为（1．50±0．21）、（1．58±0．39）mmoL／L（P〈0．05），血磷分别为（0．71±0．16）、（0．71±0．18）mmol/L（P〉0．05），血PTH升高倍数分别为（11．84±10．63）、（13．89±11．51）倍（P〉0．05）。结论 PHPT患者中CASR基因G990R多态性的分布频率与健康人群不同，R等位基因频率较高；A986S、G990R多态性与血钙、游离钙水平相关，含S或G等位基因的个体血钙及游离钙水平较低；A986S多态性与血PTH水平相关，含S等位基因的基因型的PTH水平较低。     

Agonist activation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site

Conventional nicotinic acetylcholine receptor (nAChR) agonists, such as acetylcholine, act at an extracellular "orthosteric" binding site located at the interface between two adjacent subunits. Here, we present evidence of potent activation of α7 nAChRs via an allosteric transmembrane site. Previous studies have identified a series of nAChR-positive allosteric modulators (PAMs) that lack agonist activity but are able to potentiate responses to orthosteric agonists, such as acetylcholine. It has been shown, for example, that TQS acts as a conventional α7 nAChR PAM. In contrast, we have found that a compound with close chemical similarity to TQS (4BP-TQS) is a potent allosteric agonist of α7 nAChRs. Whereas the α7 nAChR antagonist metyllycaconitine acts competitively with conventional nicotinic agonists, metyllycaconitine is a noncompetitive antagonist of 4BP-TQS. Mutation of an amino acid (M253L), located in a transmembrane cavity that has been proposed as being the binding site for PAMs, completely blocks agonist activation by 4BP-TQS. In contrast, this mutation had no significant effect on agonist activation by acetylcholine. Conversely, mutation of an amino acid located within the known orthosteric binding site (W148F) has a profound effect on agonist potency of acetylcholine (resulting in a shift of ～200-fold in the acetylcholine dose-response curve), but had little effect on the agonist dose-response curve for 4BP-TQS. Computer docking studies with an α7 homology model provides evidence that both TQS and 4BP-TQS bind within an intrasubunit transmembrane cavity. Taken together, these findings provide evidence that agonist activation of nAChRs can occur via an allosteric transmembrane site.     

Influence of combined thrombin stimulation, surface activation, and receptor occupancy on organization of GPIb/IX receptors on human platelets

Summary. Down-regulation and clearance of as many as 60–80% of GPIb/IX receptors from exposed surfaces on thrombin-activated platelets to channels of the open canalicular system (OCS) is considered to be a fundamental mechanism regulating platelet adhesivity in vitro and in vivo . The present study has combined thrombin stimulation in suspension, surface activation on formvar grids, receptor occupancy by von Willebrand factor (vWF) and exposure to anti-vWF antibody in an effort to demonstrate the removal of GPIb/IX receptors from activated cells. Individually the stimuli failed to cause any change in the frequency of GPIb/ IX receptors. Combined, the stimuli were no more effective than when each was used alone. The only way to cause GPIb/IX to move was to add anti-vWF to thrombin-activated platelets allowed to spread on formvar grids and covered with multimers of ristocetin-activated human or bovine vWF. Translocation of GPIb/IX-vWF-anti-vWF complexes from peripheral margins into caps over cell centres, however, did not clear the peripheral zone of vWF binding capacity. Exposure of capped platelets after fixation to a second incubation with vWF demonstrated as many multimers extending from the central cap to the peripheral margins as were seen on platelets exposed a single time to vWF. Antibodies to GPIb, but not to GPIIb/IIIA, prevented the second labelling by vWF. Down-regulation or clearance of GPIb/IX, in light of this study, does not appear to be a fundamental mechanism modulating platelet adhesivity.     

Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA₁) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA₁ activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA₁ domain. Substitution of amino acid residues located within the VipD–Rab5 interface prevented Rab5 binding and PLA₁ activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization.Microbial pathogens have evolved numerous ways to subvert and exploit normal host cell processes and to cause disease. Intravacuolar pathogens use specialized translocation devices such as type IV secretion systems (T4SS) to deliver virulence proteins, so-called effectors, across the bacterial and host cell membrane into the cytosol of the infected cell (1–3). Many of the translocated effectors studied to date alter cellular events such as vesicle trafficking, apoptosis, autophagy, protein ubiquitylation, or protein synthesis, among others, thereby creating conditions that support intracellular survival and replication of the microbe (4, 5). Bacteria with a nonfunctional T4SS are often avirulent and degraded along the endolysosomal pathway, thus underscoring the importance of translocated effectors for microbial pathogenesis.Although T4SS-mediated effector translocation may be a convenient way for pathogens to manipulate host cells from within the safety of their membrane-enclosed compartment, it also creates a challenging dilemma: how can the bacteria ensure that their translocated effectors reach the correct host cell target for manipulation, and how can they prevent them from indiscriminately affecting bystander organelles or proteins that may otherwise be beneficial for intracellular survival and replication of the microbe? It is reasonable to expect that regulatory mechanisms have evolved that restrain the catalytic activity of effectors. Although detailed insight into these processes is scarce, an emerging theme among effectors is that their enzymatic activity is functionally coupled to their interaction with a particular host factor. For example, SseJ from Salmonella enterica serovar Typhimurium displays glycerophospholipid-cholesterol acyltransferase activity only on binding to the active GTPases RhoA, RhoB, or RhoC (6–8). Likewise, Pseudomonas aeruginosa ExoU requires mono- or poly-ubiquitinated proteins for the activation of its phospholipase A2 (PLA₂) domain (9), whereas Yersinia YpkA exhibits kinase activity only in the presence of host cell actin (10). Exactly how binding to host ligands results in the activation of these translocated effectors remains unclear because no structural information for these protein complexes is available.VipD is a T4SS-translocated substrate of Legionella pneumophila, the causative agent of a potentially fatal pneumonia known as Legionnaires'' disease, and another example of an effector whose catalytic activity depends on the presence of a host factor (11–14). Following uptake by human alveolar macrophages, L. pneumophila translocates VipD together with more than 250 other effector proteins through its Dot/Icm T4SS into the host cell cytoplasm (15). These effectors act on numerous host processes to mediate evasion of the endolysosomal compartment and to establish a Legionella-containing vacuole (LCV) that supports bacterial growth (16). Although the precise biological role of most L. pneumophila effectors remains unclear, we recently showed that VipD is important for endosomal avoidance by LCVs. The protein localizes to endosomes presumably by binding to the small GTPases Rab5 or Rab22, key regulators of endosomal function (13, 14). Rab GTPase binding to the C-terminal domain of VipD triggers robust phospholipase A1 (PLA₁) activity within the N-terminal domain, resulting in the removal of phosphatidylinositol 3-phosphate [PI(3)P] and potentially other lipids from endosomal membranes (14). Without PI(3)P, endosomal markers such as early endosomal antigen 1 (EEA1) are lost from these membranes, most likely rendering the endosomal compartment fusion incompetent (17). L. pneumophila mutants lacking vipD are attenuated in avoiding endosomal fusion, and their LCVs acquire the endosomal marker Rab5 more frequently than LCVs containing the parental strain producing VipD (14). Thus, by coupling PLA₁ activity to Rab5 binding, the catalytic activity of VipD is directed specifically against the endosomal compartment without visibly affecting neighboring cell organelles.VipD was originally identified in a screen for L. pneumophila effectors that interfere with the vacuolar sorting pathway in yeast (11). The N-terminal half of VipD possesses high homology to patatin, a lipid acyl hydrolase present in the potato tuber (12, 13). Analogous to other patatin-like proteins, VipD harbors a conserved serine lipase motif Gly-x-Ser-x-Gly (x = any amino acid) as part of a Ser-Asp catalytic dyad that, together with two consecutive glycine residues (Asp-Gly-Gly motif), is expected to stabilize the oxyanion intermediate during the acyl chain cleavage (13). Mutation of these conserved catalytic residues in VipD results in loss of PLA₁ activity (14), confirming their role in substrate hydrolysis.The recently reported crystal structure of VipD confirmed the predicted bimodular organization (13) and, in addition, revealed a surface loop, called “lid” in other phospholipases, that shields the entry to the catalytic site. The inhibitory lid may explain why purified recombinant VipD alone exhibits little or no PLA₁ activity in vitro. However, given that binding of Rab5 or Rab22 to VipD activates the PLA₁ activity within the N-terminal region (14), we wondered if and how this binding event causes the inhibitory lid to be removed to render the active site substrate accessible.Using an integrative approach involving X-ray crystallography, molecular dynamics, biochemistry, and cellular imaging, we now deciphered at a molecular level the mechanism that stimulates the intrinsic PLA₁ activity of VipD and determined the underlying specificity for the VipD–Rab5 interaction and endosomal targeting.