功能歧化分析揭示代谢型谷氨酸受体亚家族VFTM的分化决定位点

目的：研究哺乳动物代谢型谷氨酸受体（mGluR）亚家族捕蝇夹结构模块（VFTM）的系统进化形式,确定其VFTM的功能分化决定位点。方法：通过基于系统进化的功能歧化分析,根据贝叶斯模型计算VFTM的选择性限制位点,推测功能分化决定位点。结果：mGluR亚家族VFTM的祖先通过2次基因复制,在不同的氨基酸位点进行选择性限制,形成3组不同的mGluR亚家族成员。结论：不同功能分化决定位点的选择性功能歧化塑造了mGluR亚家族VFTM的结构与功能,为进一步研究mGluR亚家族的激活机制奠定了基础。     

Are G Protein‐Coupled Receptor Heterodimers of Physiological Relevance?—Focus on Melatonin Receptors

In mammals, the circadian hormone melatonin targets two seven‐transmembrane–spanning receptors, MT₁ and MT₂, of the G protein‐coupled receptor (GPCR) super‐family. Evidence accumulated over the last 15 yrs convincingly demonstrates that GPCRs, classically considered to function as monomers, are actually organized as homodimers and heterodimerize with other GPCR family members. These dimers are formed early in the biosynthetic pathway and remain stable throughout the entire life cycle. A growing number of observations demonstrate that GPCR oligomerization may occur in native tissues and may have important consequences on receptor function. The formation of MT₁ and MT₂ homodimers and MT₁/MT₂ heterodimers has been shown in heterologous expression systems at physiological expression levels. Formation of MT₁/MT₂ heterodimers remains to be shown in native tissues but is suggested by the documented co‐expression of MT₁ and MT₂ in many melatonin‐sensitive tissues, such as the hypothalamic suprachiasmatic nuclei, retina, arteries, and adipose tissue. Considering that multiple GPCRs are expressed simultaneously in most cells, the possible engagement into heterodimeric complexes has to be considered and taken into account for the interpretation of experimental data obtained from native tissues and knockout animals.     

Stimulation of Postsynaptic and Inhibition of Presynaptic Adenylyl Cyclase Activity by Metabotropic Glutamate: Receptor Activation

Abstract: To determine the subcellular distribution of cyclic AMP-coupled metabotropic glutamate receptors (mGluRs), the effects of glutamate agonists on adenylyl cyclase activity were examined using two hippocampal membrane preparations. These were synaptosomes (SY), which are composed of presynaptic terminals, and synaptoneurosomes (SN), which are composed of both pre-and postsynaptic elements. In SY, a water-soluble analogue of forskolin (7β-forskolin) increased enzyme activity ˜ 10-fold at the highest concentration tested. The selective metabotropic receptor agonist (1S,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3 R -ACPD) inhibited enzyme activity as did glutamate and quisqualate. l -Amino-4-phosphobutanoate ( l -AP4) had no effect on enzyme activity at any concentration tested. The metabotropic receptor antagonist l -2-amino-3-phosphopropionic acid ( l -AP3) was not effective in the SY in antagonizing the agonist-induced decreases in adenylyl cyclase activity by glutamate or 1S,3 R -ACPD. It was, however, effective at antagonizing quisqualate-induced decreases in enzyme activity. In SN, at the highest concentration tested, 7β-forskolin produced a 60-fold increase in adenylyl cyclase activity. As was observed in SY, glutamate decreased adenylyl cyclase activity in SN. In contrast, 1S,3 R -ACPD, quisqualate, and l -AP4 increased adenylyl cyclase activity. In the SN, l -AP3 was ineffective in antagonizing any agonist-induced increases (1S,3 R -ACPD, l -AP4, and quisqualate) or decreases (glutamate) in adenylyl cyclase activity. The data suggest that postsynaptic metabotropic glutamate receptor activation results in stimulation of adenylyl cyclase activity, whereas inhibition of this enzyme appears to be mediated at least partly through presynaptic mechanisms.     

Effects of Human Type 1α Metabotropic Glutamate Receptor Expression Level on Phosphoinositide and Ca2+ Signalling in an Inducible Cell Expression System

Abstract: Stable expression of the human type 1α metabotropic glutamate (mGlu1α) receptor was achieved in Chinese hamster ovary cells using an isopropyl-β- d -thiogalactoside (IPTG)-repressible expression system. Treatment of the cells with IPTG resulted in a time- and concentration-dependent induction of receptor expression. Maximal expression was obtained after treatment of the cells with 100 µ M IPTG for 20 h, leading to a marked increase in receptor immunoreactivity detected by western blot, >30-fold stimulation of ³H-labelled inositol phosphate (³H-InsP) production, and a robust increase in intracellular calcium concentration in single cells after stimulation with 20 µ M quisqualate. The basal level of ³H-InsP accumulation in cells induced with IPTG was increased by two- to threefold as compared with control cells; however, this basal activity was found to be dependent on glutamate released by the cells into the incubation medium. Following IPTG treatment, stable expression of the mGlu1α receptor was maintained for at least 1 week. Taken together, these results clearly indicate the advantages of working with an inducible expression system when studying the biochemical and pharmacological properties of the human mGlu1α receptor in transfected cells.     

Metabotropic Glutamate Receptor Agonists Potentiate Cyclic AMP Formation Induced by Forskolin or β-Adrenergic Receptor Activation in Cerebral Cortical Astrocytes in Culture

Abstract: The metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (ACPD) potentiated the accumulation of cyclic AMP induced by either β-adrenergic receptor stimulation (isoproterenol) or direct activation of adenylyl cyclase (AC) with forskolin in rat cerebral cortical astrocytes grown in a defined medium. In contrast, ACPD inhibits the cyclic AMP response in astrocytes cultured in a serum-containing medium. Pharmacological characterization indicated that a group I mGluR, of which only mGluR5 is detectable in these cells, is involved in the potentiation of cyclic AMP accumulation. Potentiation was elicited by mGluR I agonists [e.g., ( R,S )-3,5-dihydroxyphenylglycine (DHPG)], but not by mGluR II or III agonists; it was pertussis toxin resistant and abolished by procedures suppressing mGluR5 function (phorbol ester pretreatment or DHPG-induced receptor down-regulation). Nevertheless, it appears that products generated through the mGluR5 transduction pathway, such as elevated [Ca²⁺]_i or activated protein kinase C (PKC), are not involved in the potentiation as it was not influenced by either the intracellular calcium chelator BAPTA-AM or the PKC inhibitor Ro 31-8220. An inhibitor of phospholipase C, U-73122, markedly attenuated mGluR5-activated phosphoinositide hydrolysis but did not significantly affect the DHPG potentiation of the cyclic AMP response. A mechanism is proposed in which the potentiating effect on AC could be mediated by free βγ complex that is liberated after the agonist-bound mGluR5 interacts with its coupled G protein.     

趋化因子受体CXCR4b在大肠杆菌中的高效表达、增溶及纯化

膜蛋白在细胞分化、信号转导等生理活动中发挥着重要作用,然而膜蛋白结构与功能的研究却受到高质量蛋白制备的严重制约。将斑马鱼趋化因子受体CXCR4b基因克隆到pMAL-p4x表达载体中,在大肠杆菌TB1中表达麦芽糖结合蛋白(MBP)-CXCR4b融合蛋白。通过系统优化其发酵表达条件,实现了CXCR4b的过量表达。最佳表达条件为:宿主菌选用大肠杆菌TB1,TB培养基,诱导剂IPTG浓度为0.5mmol/L,诱导时机为对数中后期。通过对10种不同表面活性剂的筛选,发现DM、FC-14和Brij35等表面活性剂对CXCR4b有较好的增溶效果。利用Ni2+亲和色谱和S200凝胶色谱两步纯化,得到CXCR4b的纯度可达90%以上。圆二色谱检测显示纯化的CXCR4b呈典型的α螺旋结构。     

Ins and outs of GPCR signaling in primary cilia

Primary cilia are specialized microtubule‐based signaling organelles that convey extracellular signals into a cellular response in most vertebrate cell types. The physiological significance of primary cilia is underscored by the fact that defects in assembly or function of these organelles lead to a range of severe diseases and developmental disorders. In most cell types of the human body, signaling by primary cilia involves different G protein‐coupled receptors (GPCRs), which transmit specific signals to the cell through G proteins to regulate diverse cellular and physiological events. Here, we provide an overview of GPCR signaling in primary cilia, with main focus on the rhodopsin‐like (class A) and the smoothened/frizzled (class F) GPCRs. We describe how such receptors dynamically traffic into and out of the ciliary compartment and how they interact with other classes of ciliary GPCRs, such as class B receptors, to control ciliary function and various physiological and behavioral processes. Finally, we discuss future avenues for developing GPCR‐targeted drug strategies for the treatment of ciliopathies.     

Melanin‐Concentrating Hormone Receptor Mutations and Human Obesity: Functional Analysis

Melanin‐concentrating hormone (MCH), a neuropeptide highly expressed in the lateral hypothalamus, has an important role in the regulation of energy balance and body weight in rodents. We examined whether mutations in the two known MCH receptors might be associated with obesity‐related phenotypes in humans. Among 106 subjects with severe early onset obesity and a history of hyperphagia, we found two missense variants in MCHR1: Y181H and R248Q. Neither of these was found in 192 normal weight controls. R248Q cosegregated with obesity across two generations; family data were unavailable for Y181H. When expressed in HEK293 cells, R248Q showed no evidence of constitutive activation or ligand hypersensitivity for extracellular signal‐regulated kinase phosphorylation. In addition, R248Q showed no enhanced suppression of cAMP generation. Two common single‐nucleotide polymorphisms were found to be in linkage disequilibrium: g.‐114A>G and c.39C>T. No association between either of these single‐nucleotide polymorphisms and obesity‐related phenotypes was found among a population cohort of 541 whites. Only two rare noncoding variants were found in MCHR2. In conclusion, mutations in the MCH receptors are not commonly found in humans with severe early onset obesity. Clarification of the relationship of these variants to obesity must await study in other populations and/or in genetically modified mice.     

Glutamate Exocytosis and MARCKS Phosphorylation Are Enhanced by a Metabotropic Glutamate Receptor Coupled to a Protein Kinase C Synergistically Activated by Diacylglycerol and Arachidonic Acid

Abstract: 4-Aminopyridine evokes repetitive firing of synaptosomes and exocytosis of glutamate by inhibiting a dendrotoxin-sensitive K⁺ channel responsible for stabilizing the membrane potential. We have shown previously that activation of protein kinase C (PKC) by high concentrations of phorbol ester (4β-phorbol dibutyrate) can increase release by inhibiting a dendrotoxin-insensitive ion channel, whereas the metabotropic glutamate receptor (mGluR) agonist (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate [(1 S ,3 R )-ACPD] mimics the action of 4β-phorbol dibutyrate, but only in the presence of 2 µ M arachidonic acid (AA). In this article, we investigate the role of AA. AA plus (1 S ,3 R )-ACPD is without effect on KCl-induced glutamate exocytosis, indicating that the regulatory pathway acts upstream of the release-coupled Ca²⁺ channel or Ca²⁺-secretion coupling. Diacylglycerol concentrations are greatly enhanced by (1 S ,3 R )-ACPD alone, independently of AA, indicating that AA acts downstream of phospholipase C. Myristoylated alanine-rich C kinase substrate (MARCKS) is the major presynaptic substrate for PKC. mGluR activation by (1 S ,3 R )-ACPD enhances phosphorylation of MARCKS, but only in the presence of AA. These results strongly suggest that AA acts on presynaptic PKC synergistically with diacylglycerol generated by the phospholipase-coupled mGluR, consistent with the known behaviour of certain purified PKC isoforms. The magnitude of the effects observed in a population of rat cerebrocortical synaptosomes suggests that this is a major mechanism regulating the release of the brain's dominant excitatory neurotransmitter and supports the concept that AA, or a related compound with a similar locus of action, may in certain circumstances play a role in synaptic plasticity.     

The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons

To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC(50) concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M(1) mACh receptor responsiveness is decreased in quiescent (+tetrodotoxin) neurons and increased when synaptic activity is enhanced by blocking GABA(A) receptors with picrotoxin. The picrotoxin-mediated effect on M1 mACh receptor responsiveness was completely prevented by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor blockade. Inhibition of endogenous G protein-coupled receptor kinase 2 by transfection with the non-G(q/11)alpha-binding, catalytically-inactive (D110A,K220R)G protein-coupled receptor kinase 2 mutant, decreased the extent of M1 mACh receptor desensitization under all conditions. Pharmacological inhibition of protein kinase C (PKC) activity, or chronic phorbol ester-induced PKC down-regulation had no effect on agonist-mediated receptor desensitization in quiescent or spontaneously synaptically active neurons, but significantly decreased the extent of receptor desensitization in picrotoxin-treated neurons. MCh stimulated the translocation of diacylglycerol- sensitive eGFP-PKCepsilon, but not Ca2+/diacylglycerol-sensitive eGFP-PKCbetaII in both the absence, and presence of tetrodotoxin. Under these conditions, MCh-stimulated eGFP-myristoylated, alanine-rich C kinase substrate translocation was dependent on PKC activity, but not Ca2+/calmodulin. In contrast, picrotoxin-driven translocation of myristoylated, alanine-rich C kinase substrate was accompanied by translocation of PKCbetaII, but not PKCepsilon, and was dependent on PKC and Ca2+/calmodulin. Taken together these data suggest that the level of synaptic activity may determine the different kinases recruited to regulate M1 mACh receptor desensitization in neurons.     

应用进化踪迹及分子动力学模拟研究beta2肾上腺素受体突变活性

β2肾上腺素受体(β2adrenergic receptor,β2AR)是G蛋白耦联受体(G protein coupled receptors,GPCRs)超家族中的一员,也是研究治疗哮喘的关键药物受体靶标.采用进化踪迹(evolutionary trace,ET)方法分析肾上腺素受体家族跨膜区片段序列,识别出了44个保守的残基,然后将β2肾上腺素受体以及受体D130N活性突变体、D79N失活突变体进行分子动力学模拟,试图找出与受体不同功能状态相关的结构动力学特征.发现受体DRY motif中的D130远离R131而转向K149残基这一结构特征与受体活性高度关联,此外,从残基相互作用的变化推断出了受体helix 2,4 and 6伴随着受体活化而发生的运动.这些研究结果对进一步探索β2肾上腺素受体突变体的激活机制以及所诱发疾病的分子机理提供了依据.     

PKD, PKD2, and p38 MAPK mediate Hsp27 serine-82 phosphorylation induced by neurotensin in pancreatic cancer PANC-1 cells

It is widely recognized that Hsp27 is a downstream substrate of the p38 MAPK cascade whereas the role of PKD family members in mediating receptor-stimulated Hsp27 Ser-82 phosphorylation has not been evaluated. Here, we show that neurotensin induced a rapid and striking increase in Hsp27 Ser-82 phosphorylation in PANC-1 cells, which was closely correlated with stimulation of activation loop phosphorylation of PKDs and p38 MAPK Thr180/Tyr182 phosphorylation. Treatment of PANC-1 cells with either the selective PKC inhibitor GF-I or the p38 MAPK inhibitor SB202190 partially reduced neurotensin-induced Hsp27 Ser-82 phosphorylation. However, treatment of the cells with a combination of GF-I and SB202190 virtually abolished neurotensin-induced Hsp27 Ser-82 phosphorylation. Overexpression of PKD in stably transfected PANC-1 cells increased the magnitude and prolonged the duration of Hsp27 Ser-82 phosphorylation in response to neurotensin. Either PKD or PKD2 gene silencing utilizing siRNAs targeting distinct PKD or PKD2 sequences reduced neurotensin-stimulated Hsp27 Ser-82 phosphorylation, but cotransfection of siRNAs targeting both, PKD and PKD2, markedly decreased neurotensin-induced Hsp27 Ser-82 phosphorylation. Knockdown of PKD and PKD2 abolished Hsp27 phosphorylation in cells treated with SB202190. Thus, neurotensin induces Hsp27 Ser-82 phosphorylation through p38 MAPK- and PKC/PKD-dependent pathways in PANC-1 cells. Our results demonstrate, for the first time, that neurotensin induces a striking increase in Hsp27 phosphorylation on Ser-82 in PANC-1 cells through convergent p38 MAPK, PKD, and PKD2 signaling.     

Modulation of the neuronal response to ischaemia by somatostatin analogues in wild-type and knock-out mouse retinas

Somatostatin acts at five G protein-coupled receptors, sst₁-sst₅. In mouse ischaemic retinas, the over-expression of sst₂ (as in sst₁ knock-out mice) results in the reduction of cell death and glutamate release. In this study, we reported that, in wild-type retinas, somatostatin, the multireceptor ligand pasireotide and the sst₂ agonist octreotide decreased ischaemia-induced cell death and that octreotide also decreased glutamate release. In contrast, cell death was increased by blocking sst₂ with cyanamide. In sst₂ over-expressing ischaemic retinas, somatostatin analogues increased cell death, and octreotide also increased glutamate release. To explain this reversal of the anti-ischaemic effect of somatostatin agonists in the presence of sst₂ over-expression, we tested sst₂ desensitisation because of internalisation or altered receptor function. We observed that (i) sst₂ was not internalised, (ii) among G protein-coupled receptor kinases (GRKs) and regulators of G protein signalling (RGSs), GRK1 and RGS1 expression increased following ischaemia, (iii) both GRK1 and RGS1 were down-regulated by octreotide in wild-type ischaemic retinas, (iv) octreotide down-regulated GRK1 but not RGS1 in sst₂ over-expressing ischaemic retinas. These results demonstrate that sst₂ activation protects against retinal ischaemia. However, in the presence of sst₂ over-expression sst₂ is functionally desensitised by agonists, possibly because of sustained RGS1 levels.     

Regulation of Metabotropic Glutamate Receptor 7 (mGluR7) Internalization and Surface Expression by Ser/Thr Protein Phosphatase 1

The metabotropic glutamate receptor type 7 (mGluR7) is the predominant group III mGluR in the presynaptic active zone, where it serves as an autoreceptor to inhibit neurotransmitter release. Our previous studies show that PKC phosphorylation of mGluR7 on Ser-862 is a key mechanism controlling constitutive and activity-dependent surface expression of mGluR7 by regulating a competitive interaction of calmodulin and protein interacting with C kinase (PICK1). As receptor phosphorylation and dephosphorylation are tightly coordinated through the precise action of protein kinases and phosphatases, dephosphorylation by phosphatases is likely to play an active role in governing the activity-dependent or agonist-induced changes in mGluR7 receptor surface expression. In the present study, we find that the serine/threonine protein phosphatase 1 (PP1) has a crucial role in the constitutive and agonist-induced dephosphorylation of Ser-862 on mGluR7. Treatment of neurons with PP1 inhibitors leads to a robust increase in Ser-862 phosphorylation and increased surface expression of mGluR7. In addition, Ser-862 phosphorylation of both mGluR7a and mGluR7b is a target of PP1. Interestingly, agonist-induced dephosphorylation of mGluR7 is regulated by PP1, whereas NMDA-mediated activity-induced dephosphorylation is not, illustrating there are multiple signaling pathways that affect receptor phosphorylation and trafficking. Importantly, PP1γ1 regulates agonist-dependent Ser-862 dephosphorylation and surface expression of mGluR7.     

Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

Calcitonin gene‐related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor‐like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP‐CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1‐CLR ECD fusion purified as a monomer, whereas the RAMP2‐CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27‐37) and AM(37‐52) fragments were identified as the minimal ECD complex binding regions. The CGRP C‐terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C‐terminal amide group was essential for ECD binding. Alanine‐scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries.