PDZ支架蛋白PDZK1通过PDZ1与PDZ3结构域与磷脂酶Cβ2羧基末端PDZ结合模块相互作用（英文）

磷脂酶Cβ(PLCβ)在G蛋白偶联受体(GPCR)介导的细胞信号转导中发挥重要作用.通过水解磷脂酰肌醇4,5二磷酸(PIP2),磷脂酶Cβ可以产生3种重要的第二信使分子:二乙酰甘油(DAG)、三磷酸肌醇(IP3)和质子.在果蝇中,磷脂酶Cβ通过它的羧基末端盘状同源区域结合模块(PBM)与盘状同源区域(PDZ)支架蛋白—失活无后电位D蛋白(INAD)相互作用,从而调节果蝇的光信号传导.在哺乳动物中,磷脂酶Cβ家族有4个亚型,每1个亚型的羧基末端都有1个典型的盘状同源区域结合模块.这一结构特点提示我们,磷脂酶Cβ可能通过其羧基末端的盘状同源区域结合模块与盘状同源区域支架蛋白相互作用,进而调节它们自身的细胞定位和功能.然而,目前仍对哺乳动物磷脂酶Cβ家族的盘状同源区域结合蛋白知之甚少.本文运用分析型凝胶过滤和等温滴定量热技术,系统地研究了不同磷脂酶Cβ亚型的羧基末端盘状同源区域结合模块与不同盘状同源区域蛋白质的结合.结果表明,磷脂酶Cβ2的羧基末端盘状同源区域结合模块,可以特异地与含有4个盘状同源区域的支架蛋白—盘状同源区域蛋白1(PDZK1)以2∶1的方式相互结合.进一步的测定显示,磷脂酶Cβ2羧基末端盘状同源区域结合模块在盘状同源区域蛋白1上的结合位点为第1和第3个盘状同源区域,而它们与磷脂酶Cβ2的解离常数分别为11.8±3.4μmol/L和33.3±8.7μmol/L.     

PICK1中N端酸性区域对其PDZ结构域脂质结合能力的调节

蛋白激酶Cα相互作用蛋白1(protein interacting with Cα kinase 1, PICK1)是衔接膜上受体和蛋白激酶Cα的重要蛋白.利用荧光光谱结合定点突变技术 、蛋白与脂质覆盖法等方法,分析了PICK1蛋白N末端区域几个酸性氨基酸残基对PDZ 结构域与膜脂结合的影响,以及钙离子结合N末端酸性区域对PDZ脂结合能力的调节. 结果显示, 带有上游酸性区域的PDZ结构域（NPDZ）的脂质结合能力仅相当PDZ结构 域的15%,相比单独的PDZ结构域与脂质的解离常数Kd(PDZ)为1.58×103 μg·L-1, NPDZ与脂质解离常数Kd(NPDZ)为3.3×104μg·L-1,其中在N末端酸性残基中D8与 D12两个天冬氨酸是影响脂质结合能力减弱的关键残基,若将二者分别突变为丙氨酸 后,NPDZ与脂质的解离常数分别为：Kd (D8/A)=4.42×103μg·L-1;Kd (D12/A) =1.73×103μg·L-1接近于PDZ结构域与脂质结合能力;钙离子会增强NPDZ脂结合能力,当钙离子浓度达到30 μmol/L时,NPDZ的脂结合能力提高2.3倍,但只相当于PDZ的50% 的结合能力.     

重组人磷脂酶D1/腺病毒表达载体的构建与表达

将磷脂酶D1基因及其功能缺陷点突变基因从真核表达载体pCGNPLD1亚克隆至带有绿色荧光标记蛋白的穿梭质粒pAdTrackCMV中;再与腺病毒骨架载体一起在大肠杆菌BJ5183中进行同源重组;阳性重组子经PacⅠ线性化后,转染入病毒组装细胞系293细胞,成功构建磷脂酰胆碱专一性磷脂酶D1重组腺病毒; 并用该病毒颗粒感染嗜铬细胞瘤细胞PC12细胞,高效表达磷脂酶D1蛋白。证明大蛋白基因,如磷脂酶D1基因的同源重组腺病毒表达构建切实可行,为研究其在细胞内的生理功能提供了有力工具。     

PDZ结构域的结构特点及其识别特异性配体的机制

PDZ结构域作为介导蛋白质之间相互作用的重要结构域之一,参与到细胞内运输、离子通道、以及各种信号传导通路等多种生物学过程.PDZ结构域是由80~100个氨基酸组成的小的球状结构域,对某些多PDZ结构域蛋白来说,需要一前一后形成串联体才能正确折叠.另外,PDZ结构域相互之间也可以形成同源或异源二聚体.这些PDZ结构域的突出特点是能特异性地识别配体靶蛋白C末端短的氨基酸序列,但有些也能识别靶蛋白的内部β发夹结构.而一些支架蛋白的PDZ结构域与细胞膜上脂类的相互作用则增加了其与膜的亲和性.本文简要概括了PDZ结构域的结构特点及其对配体的各种特异性识别的机制,从而为研究各种PDZ蛋白的功能提供了结构基础.     

δ阿片肽受体分子药理学

目前已成功地克隆出δ、μ、κ阿片受体 ,均属Ｇ蛋白偶联受体 ,有 6 5 %同源序列 ,仅 35 %序列决定其特异性。阿片受体最大的同源区是跨膜区 (ｔｒａｎｓｍｅｍｂｒａｎｅ ,ＴＭ)和细胞内环 ,变化最大的区域在细胞外环及其氨基、羧基末端。近年来应用反义核酸技术、基因剔除、构建嵌合受体、基因定位突变、截短或缺失氨基酸突变等方法对阿片受体的结构和功能的研究取得了新进展。1 .内源性与克隆δ阿片受体δ阿片受体广泛分布于脑内 ,但在不同的脑区其分布密度不同。体内药理学实验证明 ,δ阿片受体有两种亚型δ1和δ2 [1] ,但是其亚型没被…     

PGC-1β和SREBP-1c在猪脂肪细胞分化过程中的相互作用

为研究过氧化物酶体增殖物激活受体γ辅激活因子1β(PGC-1β)与SREBP-1c在猪前体脂肪细胞分化过程中的表达规律及其相互作用,分析二者功能上的联系,采用Western 印迹及细胞免疫荧光技术检测PGC-1β与SREBP-1c在猪脂肪细胞分化过程中的表达,shRNA干扰和免疫共沉淀技术分别探讨了PGC-1β对SREBP-1c的调节作用及2种蛋白质在体内的结合活性.结果显示,PGC-1β与SREBP-1c 蛋白的表达均随猪脂肪细胞分化逐渐增加,且在分化细胞的核和胞浆中均有分布. 干扰PGC-1β显著下调了SREBP-1c和脂肪细胞分化标记基因C/EBPα的表达(P<0.05),同时降低了细胞内甘油三酯的积累.免疫共沉淀证明,PGC-1β与SREBP-1c蛋白在猪脂肪细胞分化过程中存在结合作用. 以上结果表明,PGC-1β能够促进猪脂肪细胞分化并对SREBP-1c有调节和结合作用,推测二者的结合可能与其对脂肪细胞的分化调节机制相关,将对PGC-1β调控脂肪细胞分化的功能和机理研究提供新途径.     

利用随机多肽文库研究ZO-1中PDZ3结构域的配体结合特点

建立一种研究PDZ结构域配体结合特点的简单方法 .利用酵母双杂交技术从随机多肽文库中寻找所有可能与ZO 1中PDZ3结构域结合的C末端序列 ,从现有蛋白质数据库中检索所有具有该C末端蛋白 .利用液体培养物 β 半乳糖苷酶检测实验 ,比较文库中筛选的C末端序列和已知的PDZ3结构域结合配体———JAM的C末端 (SFLV)与PDZ3结构域结合的强弱 .共筛选到 3个阳性克隆 ,其C末端序列分别为 LGWV、 LVWV和 DEWV .前 2者属于第二类PDZ结构域 ,后者属于第三类 .蛋白质数据库检索结果表明 ,有多个蛋白质具有 LGWV、 LVWV末端 ,没有检索到任何具有 DEWV末端的蛋白质 .结合强度实验结果表明 ,它们与PDZ3结构域结合强度依次为 DEWV > LGWV > LVWV > SFLV ,说明筛选的 3个C末端除了反映ZO 1中PDZ3结构域可能的潜在结合配体外 ,也有可能成为JAM蛋白阻断性试剂甚至药物的重要组成部分之一 .利用随机多肽文库 ,可以尽可能寻找所有可能与PDZ结构域结合的C末端序列 ,大大提高了基因文库筛选的效率     

KLFs对珠蛋白基因表达和红系分化的调控作用

Krüppel样因子(Krüppel-like factors, KLFs)是一组与真核基因转录调控密切相关的锌指蛋白.KLFs高度保守的羧基末端含3个串联的Cys2His2型锌指结构,用于结合GC盒和CACCC盒等DNA序列. 红细胞中特异表达的珠蛋白基因和许多红系调控因子中都含有CACCC盒.已有研究发现,多个KLFs通过结合CACCC盒参与调控珠蛋白基因表达和红系分化,例如,KLF1通过结合β-珠蛋白启动子和位点控制区(locus control region, LCR),促进β-珠蛋白的表达、γ-向β-珠蛋白基因的转换和红系分化;KLF2、KLF11和KLF13分别促进ε-和γ-珠蛋白基因的表达;KLF4促进α-和γ-珠蛋白基因的表达;KLF3和KLF8则抑制ε-和γ-珠蛋白基因的表达. 本文综述了KLFs调控珠蛋白基因表达和红系分化的研究进展.     

C型产气荚膜梭菌α、β_1毒素基因的融合

利用PCR技术,从C型产气荚膜梭菌染色体DNA中扩增出α和β1毒素基因,通过分离、纯化、内切酶酶切、连接和转化,构建了含αβ1融合基因表达质粒重组菌株BL21(DE3)(pETXAB1)。经酶切鉴定和核苷酸序列测定证实,构建的重组质粒pETXAB1含有αβ1融合基因,且基因序列和阅读框架均正确。经ELISA检测,重组菌株表达的αβ1融合蛋白能够被α、β1毒素抗体识别。免疫实验结果表明,αβ1融合蛋白免疫的小鼠可以抵抗1MLD的C型产气荚膜梭菌C5944毒素攻击,表明构建的重组菌株可以作为预防仔猪红痢基因工程亚单位苗的候选菌株。     

人类β-1,4-半乳糖苷转移酶基因家族中1个新成员的克隆与表达谱分析

应用从EST出发克隆基因家族新成员的策略 ,在人类睾丸组织cDNA文库中分离出 1个新的全长cDNA片段 ,它编码的蛋白产物与 (UDP 半乳糖苷 :N 乙酰基葡萄糖胺 )半乳糖苷转移酶 (GalT)有较高的同源度 .分析表明 ,在其 2 173bp的序列中含有 1个长 10 3 2bp的开放读框 ,编码 3 44个氨基酸 .基于该基因与半乳糖苷转移酶基因家族各成员的同源关系 ,尤其是与G .gallus β 1,4 半乳糖苷转移酶Ⅰ型(CKⅠ )的关系密切 ,这个基因的蛋白产物被命名为人类 β 1,4 半乳糖苷转移酶同源蛋白Ⅰ型 .Northern杂交发现它在被检测的人体 16种组织中均有表达 ,但丰度各异 .通过与含人单条染色体的人 /啮齿类的杂种细胞DNA进行Southern杂交 ,证明该基因位于 3号染色体上 .     

Domain-swapped dimerization of ZO-1 PDZ2 generates specific and regulatory connexin43-binding sites

PDZ domain scaffold proteins are capable of assembling macromolecular protein complexes in diverse cellular processes through PDZ-mediated binding to a short peptide fragment at the carboxyl tail of target proteins. How each PDZ domain specifically recognizes its target protein(s) remains a major conceptual question, as at least a few out of the several hundred PDZ domains in each eukaryotic genome share overlapping binding properties with any given target protein. Here, we show that the domain-swapped dimerization of zonula occludens-1 PDZ2 generates a distinct interface that functions together with the well-separated canonical carboxyl tail-binding pocket in each PDZ unit in binding to connexin43 (Cx43). We further demonstrate that the charge-charge interaction network formed by residues in the PDZ dimer interface and upstream residues of the Cx43 peptide not only provides the unprecedented interaction specificity for the complex but may also function as a phosphorylation-mediated regulatory switch for the dynamics of the Cx43 gap junctions. Finally, we provide evidence that such domain-swapped dimer assembly also occurs in other PDZ domain scaffold proteins. Therefore, our findings present a new paradigm for understanding how some PDZ domain proteins specifically bind to and regulate the functions of their target proteins.     

Supramodular structure and synergistic target binding of the N-terminal tandem PDZ domains of PSD-95

PDZ domain proteins play critical roles in binding, clustering and subcellular targeting of membrane receptors and ion channels. PDZ domains in multi-PDZ proteins often are arranged in groups with highly conserved spacing and intervening sequences; however, the functional significance of such tandem arrangements of PDZs is unclear. We have solved the three-dimensional structure of the first two PDZ domains of postsynaptic density protein-95 (PSD-95 PDZ1 and PDZ2), which are closely linked to each other in the PSD-95 family of scaffold proteins. The two PDZs have limited freedom of rotation and their C-terminal peptide-binding grooves are aligned with each other with an orientation preference for binding to pairs of C termini extending in the same direction. Increasing the spacing between PDZ1 and PDZ2 resulted in decreased binding between PDZ12 and its dimeric targets. The same mutation impaired the functional ability of PSD-95 to cluster Kv1.4 potassium channels in heterologous cells. The data presented provide a molecular basis for preferential binding of PSD-95 to multimeric membrane proteins with appropriate C-terminal sequences.     

Using a collection of MUPP1 domains to investigate the similarities of neurotransmitter transporters C-terminal PDZ motifs

A ubiquitous feature of neurotransmitter transporters is the presence of short C-terminal PDZ binding motifs acting as important trafficking elements. Depending on their very C-terminal sequences, PDZ binding motifs are usually divided into at least three groups; however this classification has recently been questioned. To introduce a 3D aspect into transporter’s PDZ motif similarities, we compared their interactions with the natural collection of all 13 PDZ domains of the largest PDZ binding protein MUPP1. The GABA, glycine and serotonin transporters showed unique binding preferences scattered over one or several MUPP1 domains. On the contrary, the dopamine and norepinephrine transporter PDZ motifs did not show any significant affinity to MUPP1 domains. Interestingly, despite their terminal sequence diversity all three GABA transporter PDZ motifs interacted with MUPP1 domain 7. These results indicate that similarities in binding schemes of individual transporter groups might exist. Results also suggest the existence of variable PDZ binding modes, allowing several transporters to interact with identical PDZ domains and potentially share interaction partners in vivo.     

NHE3 Regulatory Factor 1 (NHERF1) Modulates Intestinal Sodium-dependent Phosphate Transporter (NaPi-2b) Expression in Apical Microvilli

P_i uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary P_i but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2_BBE cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1^−/− mice, but not PDZK1^−/− mice, had a diminished adaptation of NaPi-2b expression in response to a low P_i diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.     

Structure of PICK1 and other PDZ domains obtained with the help of self-binding C-terminal extensions

PDZ domains are protein-protein interaction modules that generally bind to the C termini of their target proteins. The C-terminal four amino acids of a prospective binding partner of a PDZ domain are typically the determinants of binding specificity. In an effort to determine the structures of a number of PDZ domains we have included appropriate four residue extensions on the C termini of PDZ domain truncation mutants, designed for self-binding. Multiple truncations of each PDZ domain were generated. The four residue extensions, which represent known specificity sequences of the target PDZ domains and cover both class I and II motifs, form intermolecular contacts in the expected manner for the interactions of PDZ domains with protein C termini for both classes. We present the structures of eight unique PDZ domains crystallized using this approach and focus on four which provide information on selectivity (PICK1 and the third PDZ domain of DLG2), binding site flexibility (the third PDZ domain of MPDZ), and peptide-domain interactions (MPDZ 12th PDZ domain). Analysis of our results shows a clear improvement in the chances of obtaining PDZ domain crystals by using this approach compared to similar truncations of the PDZ domains without the C-terminal four residue extensions.     

In Vitro and in Vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B,Type I (SR-BI), to the PDZ1 Domain of Its Adaptor Protein PDZK1

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys¹⁴-Xaa₄-Asn¹⁹-Tyr-Gly-Phe-Phe-Leu²⁴), and measured their binding affinity for a 7-residue peptide corresponding to the C terminus of SR-BI (⁵⁰³VLQEAKL⁵⁰⁹). The Y20A and G21Y substitutions abrogated all binding activity. Surprisingly, binding affinities (K_d) of the K14A and F22A mutants were 3.2 and 4.0 μm, respectively, similar to 2.6 μm measured for the WT PDZ1. To understand these findings, we determined the high resolution structure of WT PDZ1 bound to a 5-residue sequence from the C-terminal SR-BI (⁵⁰⁵QEAKL⁵⁰⁹) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10–20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.     

D‐AKAP2:PKA RII:PDZK1 ternary complex structure: Insights from the nucleation of a polyvalent scaffold

A‐kinase anchoring proteins (AKAPs) regulate cAMP‐dependent protein kinase (PKA) signaling in space and time. Dual‐specific AKAP2 (D‐AKAP2/AKAP10) binds with high affinity to both RI and RII regulatory subunits of PKA and is anchored to transporters through PDZ domain proteins. Here, we describe a structure of D‐AKAP2 in complex with two interacting partners and the exact mechanism by which a segment that on its own is disordered presents an α‐helix to PKA and a β‐strand to PDZK1. These two motifs nucleate a polyvalent scaffold and show how PKA signaling is linked to the regulation of transporters. Formation of the D‐AKAP2: PKA binary complex is an important first step for high affinity interaction with PDZK1, and the structure reveals important clues toward understanding this phenomenon. In contrast to many other AKAPs, D‐AKAP2 does not interact directly with the membrane protein. Instead, the interaction is facilitated by the C‐terminus of D‐AKAP2, which contains two binding motifs—the D‐AKAP2_AKB and the PDZ motif—that are joined by a short linker and only become ordered upon binding to their respective partner signaling proteins. The D‐AKAP2_AKB binds to the D/D domain of the R‐subunit and the C‐terminal PDZ motif binds to a PDZ domain (from PDZK1) that serves as a bridging protein to the transporter. This structure also provides insights into the fundamental question of why D‐AKAP2 would exhibit a differential mode of binding to the two PKA isoforms.     

Structure determination and ligand interactions of the PDZ2b domain of PTP-Bas (hPTP1E): splicing-induced modulation of ligand specificity

Two versions of the PDZ2 domain of the protein tyrosine phosphatase PTP-Bas/human PTP-BL are generated by alternative splicing. The domains differ by the insertion of five amino acid residues and their affinity to the tumour suppressor protein APC. Whereas PDZ2a is able to bind APC in the nanomolar range, PDZ2b shows no apparent interaction with APC. Here the solution structure of the splicing variant of PDZ2 with the insertion has been determined using 2D and 3D heteronuclear NMR experiments. The structural reason for the changed binding specificity is the reorientation of the loop with extra five amino acid residues, which folds back onto beta-strands two and three. In addition the side-chain of Lys32 closes the binding site of the APC binding protein and the two helices, especially alpha-helix 2, change their relative position to the protein core. Consecutively, the binding site is sterically no longer fully accessible. From the NMR-titration studies with a C-terminal APC-peptide the affinity of the peptide with the protein can be estimated as 540(+/-40)microM. The binding site encompasses part of the analogous binding site of PDZ2a as already described previously, yet specific interaction sites are abolished by the insertion of amino acids in PDZ2b. As shown by high-affinity chromatography, GST-PDZ2b and GST-PDZ2a bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles with a dissociation constant K(D) of 21 microM and 55 microM, respectively. In line with these data PDZ2b binds isolated, dissolved PIP(2) and PIP(3) (phosphatidylinositol 3,4,5-trisphosphate) molecules specifically with a lower K(D) of 230(+/-20)microM as detected by NMR spectroscopy. The binding site could be located by our studies and involves the residues Ile24, Val26, Val70, Asn71, Gly77, Ala78, Glu85, Arg88, Gly91 and Gln92. PIP(2) and PIP(3) binding takes place in the groove of the PDZ domain that is normally part of the APC binding site.     

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.     

C-terminal phosphorylation of MRP2 modulates its interaction with PDZ proteins

MRP2, a member of the ABC protein superfamily, functions as an ATP-dependent export pump for anionic conjugates in the apical membranes of epithelial cells. It has been reported that the trafficking of MRP2 is modulated by PKC. Adjacent to the C-terminal PDZ binding motif, which may be involved in the targeting of MRP2, we found a potential PKC phosphorylation site (Ser(1542)). Therefore, we examined the interaction of MRP2 and its phosphorylation-mimicking mutants with different PDZ proteins (EBP50, E3KARP, PDZK1, IKEPP, beta2-syntrophin, and SAP-97). The binding of these PDZ proteins to CFTR and ABCA1, other ABC proteins, possessing PDZ binding motif, was also studied. We observed a strong binding of apically localized PDZ proteins to both MRP2 and CFTR, whereas beta2-syntrophin exhibited binding only to ABCA1. The phosphorylation-mimicking MRP2 mutant and a phosphorylated C-terminal MRP2 peptide showed significantly increased binding to IKEPP, EBP50, and both individual PDZ domains of EBP50. Our results suggest that phosphorylation of the MRP2 PDZ binding motif has a profound effect on the PDZ binding of MRP2.