The intracellular carboxyl terminal domain of Vangl proteins contains plasma membrane targeting signals

Vangl1 and Vangl2 are integral membrane proteins that play a critical role in establishing planar cell polarity (PCP) in epithelial cells and are required for convergent extension (CE) movements during embryogenesis. Their proper targeting to the plasma membrane (PM) is required for function. We created discrete deletions at the amino and carboxy termini of Vangl1 and monitored the effect of the mutations on PM targeting in Madin–Darby canine kidney cells. Our results show that the Vangl1 amino terminus lacks PM targeting determinants, and these are restricted to the carboxy terminus, including the predicted PDZBM motif at the C‐terminus.     

Zinc binding site in PICK1 is dominantly located at the CPC motif of its PDZ domain

PICK1 ( p rotein i nteracting with C k inase 1) containing a PDZ domain, a BAR domain, and two short acidic regions is as an adaptor protein that plays an important role in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor trafficking, cell morphology and migration, as well as in some diseases such as cancer, schizophrenia and pain. To better understand the physiological function of PICK1, we expressed the recombinant PICK1 and its truncated mutants in E.coli, and measured their zinc binding properties by fluorescence and competition assay. It is shown that PICK1 has one Zn²⁺-binding site. The Zn²⁺-binding properties of PICK1 are not appreciably affected after the removal of BARC domain (involving BAR domain and C-terminal acidic region). Deleting the N-terminal acidic region of NPDZ domain (involving PDZ domain and N-terminal acidic region) in PICK1 impairs its Zn²⁺-binding capacity.The mutation of the CPC (Cys-Pro-Cys) motif in the PDZ domain of PICK1 abolishes the ability of Zn²⁺-binding. In addition, Zn²⁺ can enhance the lipid-binding ability of PDZ domain as observed in both protein-lipid overlay assay and fluorescence analysis. The results presented in this report suggested that Zn²⁺ plays a regulatory role in the trafficking of PICK1 from the cytoplasm to cell membrane.     

The glutamate transporter GLT1b interacts with the scaffold protein PSD-95

The glutamate transporter (GLT1) regulates glutamate concentrations in glutamatergic synapses and it is expressed in at least two isoforms, GLT1a and GLT1b. In this work, we show that the C-terminus of GLT1b is able to interact with the PDZ domains of a number of proteins. Notably, one of them might be the scaffold protein post-synaptic density (PSD-95). GLT1b formed co-immunoprecipitable complexes with PSD-95 in solubilizated rat brain extracts, complexes that also contained NMDA receptors. Co-transfection of GLT1b, PSD-95, and NMDA receptor subunits in heterologous expression systems recapitulated in vitro the interactions among these proteins that had been observed in the rat brain extracts and revealed the importance of the GLT1b C-terminal PDZ binding motif in tethering this transporter to PSD-95. Significantly, co-expression of GLT1b and PSD-95 increased the V _max of the transporter by decreasing the rate of GLT1b endocytosis. Moreover, GLT1b transfected into primary cultured neurons or glia formed protein clusters that co-localized with co-transfected PSD-95, clusters that in these neurons accumulated preferentially in dendritic spines. We hypothesize that the GLT1b/PSD-95 interaction, characterized here in vitro , might anchor this transporter close to the post-synaptic glutamate receptors, thereby permitting the fine regulation of glutamate concentrations in this microenvironment. This tight association might also facilitate the regulation of GLT1b through the signaling pathways initiated by the activation of glutamate receptors.     

Actin cytoskeleton-dependent dynamics of the human serotonin1A receptor correlates with receptor signaling

Analyzing the dynamics of membrane proteins in the context of cellular signaling represents a challenging problem in contemporary cell biology. Lateral diffusion of lipids and proteins in the cell membrane is known to be influenced by the cytoskeleton. In this work, we explored the role of the actin cytoskeleton on the mobility of the serotonin_1A (5-HT_1A) receptor, stably expressed in CHO cells, and its implications in signaling. FRAP analysis of 5-HT_1AR-EYFP shows that destabilization of the actin cytoskeleton induced by either CD or elevation of cAMP levels mediated by forskolin results in an increase in the mobile fraction of the receptor. The increase in the mobile fraction is accompanied by a corresponding increase in the signaling efficiency of the receptor. Interestingly, with increasing concentrations of CD used, the increase in the mobile fraction exhibited a correlation of ∼0.95 with the efficiency in ligand-mediated signaling of the receptor. Radioligand binding and G-protein coupling of the receptor were found to be unaffected upon treatment with CD. Our results suggest that signaling by the serotonin_1A receptor is correlated with receptor mobility, implying thereby that the actin cytoskeleton could play a regulatory role in receptor signaling. These results may have potential significance in the context of signaling by GPCRs in general and in the understanding of GPCR-cytoskeleton interactions with respect to receptor signaling in particular.     

Drosophila Lin-7 is a component of the Crumbs complex in epithelia and photoreceptor cells and prevents light-induced retinal degeneration

The Drosophila Crumbs protein complex is required to maintain epithelial cell polarity in the embryo, to ensure proper morphogenesis of photoreceptor cells and to prevent light-dependent retinal degeneration. In Drosophila, the core components of the complex are the transmembrane protein Crumbs, the membrane-associated guanylate kinase (MAGUK) Stardust and the scaffolding protein DPATJ. The composition of the complex and some of its functions are conserved in mammalian epithelial and photoreceptor cells. Here, we report that Drosophila Lin-7, a scaffolding protein with one Lin-2/Lin-7 (L27) domain and one PSD-95/Dlg/ZO-1 (PDZ) domain, is associated with the Crumbs complex in the subapical region of embryonic and follicle epithelia and at the stalk membrane of adult photoreceptor cells. DLin-7 loss-of-function mutants are viable and fertile. While DLin-7 localization depends on Crumbs, neither Crumbs, Stardust nor DPATJ require DLin-7 for proper accumulation in the subapical region. Unlike other components of the Crumbs complex, DLin-7 is also enriched in the first optic ganglion, the lamina, where it co-localizes with Discs large, another member of the MAGUK family. In contrast to crumbs mutant photoreceptor cells, those mutant for DLin-7 do not display any morphogenetic abnormalities. Similar to crumbs mutant eyes, however, DLin-7 mutant photoreceptors undergo progressive, light-dependent degeneration. These results support the previous conclusions that the function of the Crumbs complex in cell survival is independent from its function in photoreceptor morphogenesis.     

Identification and characterization of two novel splice forms of GRIP1 in the rat brain

We cloned two novel alternatively-spliced mRNA isoforms of glutamate receptor interacting protein 1 (GRIP1) which we named GRIP1d and GRIP1e 4-7. GRIP1d is a 135 kDa, 7-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 4-PDZ-domain GRIP1c 4-7. GRIP1e 4-7 is a 75 kDa 4-PDZ-domain variant of GRIP1, containing the 12 amino acid C-terminus originally described for the 7-PDZ-domain GRIP1a/b. Northern blots indicated that GRIP1d mRNA is 5.1 kb long and abundant in brain. An antibody to the C-terminus of the 75 kDa GRIP1c 4-7 also recognized an abundant 135 kDa protein, consistent with the predicted size of GRIP1d. Similarly, an antibody to the C-terminus of the 135 kDa GRIP1a/b also recognized a low abundance 75 kDa protein, consistent with the predicted size of GRIP1e 4-7. Immunocytochemistry of hippocampal cultures and intact brain using these antibodies showed that (i) these isoforms are present in both GABAergic and glutamatergic synapses, and (ii) the isoforms co-localize in individual synapses. While GRIP1a/b isoforms are abundant in interneurons and highly concentrated in GABAergic presynaptic terminals, the isoforms recognized by the antibody to the C-terminus common to GRIP1c 4-7 and GRIP1d are much less abundant in interneurons and preferentially concentrate at the postsynaptic complex.     

Defining the minimal interacting regions of the tight junction protein MAGI-1 and HPV16 E6 oncoprotein for solution structure studies

The oncoprotein E6 produced by tumorigenic high-risk genital human papillomaviruses targets a number of cellular proteins containing PDZ domains for proteasome-mediated degradation. In particular, E6 targets the tight junction protein MAGI-1 by binding to its PDZ1 domain. Using light scattering and NMR, we explored different fragments of both the HPV16 E6 and the MAGI-1 PDZ1 domain to define the best-behaving complex for solution structure studies. We showed that the 70-residue HPV16 E6 C-terminal domain (E6C) can be efficiently substituted by a peptide spanning the 11 C-terminal residues of E6. The construct of MAGI-1 PDZ1 best suited for solution structure analysis presents a 14-residue N-terminal extension and a 26-residue C-terminal extension as compared to the construct used for the recently solved X-ray structure of a MAGI-1 PDZ1/HPV18 E6 complex. These data suggest a stabilizing role for the interdomain linker regions which separate the PDZ1 domain from its neighboring domains.     

大鼠MUPP1蛋白PDZ8结构域的生化及晶体结构特性

多重PDZ结构域蛋白1型（MUPP1）是一种存在于上皮细胞和神经细胞内含有13个PDZ结构域的重要支架蛋白.在上皮细胞中,MUPP1蛋白在紧密连接结构的形成和上皮细胞的极化过程中发挥重要作用.而在中枢神经系统中,MUPP1基因的1个提前终止突变导致了其最后12个PDZ结构域的缺失,以及严重的先天性脑积水.此外,MUPP1蛋白的表达水平与酒精依赖性和药物戒断的严重性也具有显著的相关性.因此,对MUPP1蛋白所含的PDZ结构域进行纯化和性质鉴定,将有助于深入研究MUPP1蛋白的功能和分子机制.在本文研究中,利用亲和纯化和分子筛技术,对大鼠来源的MUPP1蛋白的第8个PDZ结构域进行了表达和纯化.多角度激光光散射的数据表明: MUPP1-PDZ8结构域在溶液中为单体,分子量为16.4 kD.圆二色谱结果表明,MUPP1-PDZ8结构域具有较好的二级结构折叠,测得其熔解温度为71.6摄氏度,暗示该PDZ结构域在溶液中非常稳定.最后,MUPP1-PDZ8结构域的晶体结构显示,该结构域属于I 型PDZ 结构域,包含3个α螺旋和6个β折叠.其中GLGL模块、β折叠B上的1 351位亮氨酸,以及α螺旋B上的1 405位异亮氨酸/1 398位组氨酸形成的PDZ结合口袋,可以特异性地与其目标蛋白质的羧基末端相结合.综上所述,本文的研究提供了MUPP1-PDZ8结构域的生化特性,以及该结构域与其目标蛋白质相互作用的分子机制,这将为MUPP1蛋白的功能研究提供生物化学与结构生物学的理论基础.     

Phosphatidylinositol 5-phosphate 4-kinase type II beta is required for vitamin D receptor-dependent E-cadherin expression in SW480 cells

Numerous epidemiological data indicate that vitamin D receptor (VDR) signaling induced by its ligand or active metabolite 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) has anti-cancer activity in several colon cancers. 1α,25(OH)₂D₃ induces the epithelial differentiation of SW480 colon cancer cells expressing VDR (SW480-ADH) by upregulating E-cadherin expression; however, its precise mechanism remains unknown. We found that phosphatidylinositol-5-phosphate 4-kinase type II beta (PIPKIIβ) but not PIPKIIα is required for VDR-mediated E-cadherin induction in SW480-ADH cells. The syntenin-2 postsynaptic density protein/disc large/zona occludens (PDZ) domain and pleckstrin homology domain of phospholipase C-delta1 (PLCδ1 PHD) possess high affinity for phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂) mainly localized to the nucleus and plasma membrane, respectively. The expression of syntenin-2 PDZ but not PLCδ1 PHD inhibited 1α,25(OH)₂D₃-induced E-cadherin upregulation, suggesting that nuclear PI(4,5)P₂ production mediates E-cadherin expression through PIPKIIβ in a VDR-dependent manner. PIPKIIβ is also involved in the suppression of the cell motility induced by 1α,25(OH)₂D₃. These results indicate that PIPKIIβ-mediated PI(4,5)P₂ signaling is important for E-cadherin upregulation and inhibition of cellular motility induced by VDR activation.     

Surface plasmon resonance analysis of the binding of high‐risk mucosal HPV E6 oncoproteins to the PDZ1 domain of the tight junction protein MAGI‐1

The E6 oncoproteins from high‐risk mucosal human papillomavirus (HPV) induce cervical cancer via two major activities, the binding and the degradation of the p53 protein and PDZ domain‐containing proteins. Human MAGI‐1 is a multi‐PDZ domain protein implicated into protein complex assembly at cell–cell contacts. High‐risk mucosal HPV E6 proteins interact with the PDZ1 domain of MAGI‐1 via a C‐terminal consensus binding motif. Here, we developed a medium throughput protocol to accurately measure by surface plasmon resonance affinity constants of protein domains binding to peptidic sequences produced as recombinant fusions to the glutathione‐S‐transferase (GST). This approach was applied to measure the binding of MAGI‐1 PDZ1 to the C‐termini of viral or cellular proteins. Both high‐risk mucosal HPV E6 C‐terminal peptides and cellular partners of MAGI‐1 PDZ1 bind to MAGI‐1 PDZ1 with comparable dissociation constants in the micromolar range. MAGI‐1 PDZ1 shows a preference for C‐termini with a valine at position 0 and a negative charge at position ?3, confirming previous studies performed with HPV18 E6. A detailed combined analysis via site‐directed mutagenesis of the HPV16 C‐terminal peptide and PDZ1 indicated that interactions mediated by charged residues upstream the PDZ‐binding motif strongly contribute to binding selectivity of this interaction. In addition, our work highlighted the K₄₉₉ residue of MAGI‐1 as a novel determinant of binding specificity. Finally, we showed that MAGI‐1 PDZ1 also binds to the C‐termini of LPP and Tax proteins, which were already known to bind to PDZ proteins but not to MAGI‐1. Copyright © 2010 John Wiley & Sons, Ltd.