Structure and function of WD40 domain proteins

The WD40 domain exhibits a β-propeller architecture, often comprising seven blades. The WD40 domain is one of the most abundant domains and also among the top interacting domains in eukaryotic genomes. In this review, we will discuss the identification, definition and architecture of the WD40 domains. WD40 domain proteins are involved in a large variety of cellular processes, in which WD40 domains function as a protein-protein or protein-DNA interaction platform. WD40 domain mediates molecular recognition events mainly through the smaller top surface, but also through the bottom surface and sides. So far, no WD40 domain has been found to display enzymatic activity. We will also discuss the different binding modes exhibited by the large versatile family of WD40 domain proteins. In the last part of this review, we will discuss how post-translational modifications are recognized by WD40 domain proteins.     

Differences in performance between trained and untrained subjects during a 30-s sprint test in a wheelchair ergometer

To compare physiological responses and propulsion technique of able bodied subjects with no prior experience of wheelchairs (AB) and wheelchair dependent subjects (WD), ten AB and nine WD performed a 30-s sprint test in a wheelchair ergometer. The WD had spinal cord injuries with a lesion at T8 or lower. The WD and AB did not show significantly different physiological responses. The power values averaged for the right wheel over the 30 s of the test were 50.2 (SD 14.7) W and 48.0 (SD 4.4) W for WD and AB, respectively. No significant differences in torque application could be discerned, although WD subjects seemed to have a more flattened torque curve with a smaller negative deflection at the beginning of the push. The WD applied a significantly higher horizontal propulsive force to the handrims but did not apply force more effectively. The percentages of effective force to total propulsive force were 61 (SD 16)% for WD and 57 (SD 4)% for AB. With regard to the kinematic parameters, AB followed the handrims significantly longer than WD (end angle AB 65°, WD 44°), started the push phase with their arms more in retroflexion and flexed their trunks further forward. The AB did however show a movement pattern comparable to that of wheelchair athletes measured in a comparable experiment. It could not be decided conclusively that inexperience in wheelchair propulsion led to a less effective propulsion technique. Despite the selection of WD with respect to lesion level, interindividual differences in terms of level of training may have been responsible for the absence of significant results.     

Genome-Wide Investigation and Expression Analyses of WD40 Protein Family in the Model Plant Foxtail Millet (Setaria italica L.)

WD40 proteins play a crucial role in diverse protein-protein interactions by acting as scaffolding molecules and thus assisting in the proper activity of proteins. Hence, systematic characterization and expression profiling of these WD40 genes in foxtail millet would enable us to understand the networks of WD40 proteins and their biological processes and gene functions. In the present study, a genome-wide survey was conducted and 225 potential WD40 genes were identified. Phylogenetic analysis categorized the WD40 proteins into 5 distinct sub-families (I–V). Gene Ontology annotation revealed the biological roles of the WD40 proteins along with its cellular components and molecular functions. In silico comparative mapping with sorghum, maize and rice demonstrated the orthologous relationships and chromosomal rearrangements including duplication, inversion and deletion of WD40 genes. Estimation of synonymous and non-synonymous substitution rates revealed its evolutionary significance in terms of gene-duplication and divergence. Expression profiling against abiotic stresses provided novel insights into specific and/or overlapping expression patterns of SiWD40 genes. Homology modeling enabled three-dimensional structure prediction was performed to understand the molecular functions of WD40 proteins. Although, recent findings had shown the importance of WD40 domains in acting as hubs for cellular networks during many biological processes, it has invited a lesser research attention unlike other common domains. Being a most promiscuous interactors, WD40 domains are versatile in mediating critical cellular functions and hence this genome-wide study especially in the model crop foxtail millet would serve as a blue-print for functional characterization of WD40s in millets and bioenergy grass species. In addition, the present analyses would also assist the research community in choosing the candidate WD40s for comprehensive studies towards crop improvement of millets and biofuel grasses.     

Relationships between nerves and myofibroblasts during cutaneous wound healing in the developing rat

Contraction of skin excision wounds is affected by age and the presence of peripheral nerves. The present study examined relationships between peripheral innervation, wound contractile cells, and rate of wound closure to determine whether these are altered during development. Full-thickness 4-mm-diameter circular flaps were excised from the interscapular skin of rats on postnatal day (PND) 5, PND 12, or PND 60. Wounds of PND 5 and PND 12 rats contracted 45% between post-wound days (WD) 3 and 5 and more slowly thereafter, with a scar 9-14% of the original wound size by WD 21. In contrast, PND 60 wounds contracted only 22% between WD 3 and 5, and the residual scar at WD 21 was 40% of the original wound size. In younger rats, alpha-smooth muscle actin-immunoreactive myofibroblasts first appeared on WD 2 and attained maximum density at WD 5. Innervation, as assessed by protein gene product 9.5 immunoreactivity, appeared by WD 3 and increased rapidly through WD 7 in younger rats. In PND 60 wounds, myofibroblasts did not appear until WD 5 and did not attain a maximum until day 10. Nerve ingrowth was not significant until WD 10 and was depressed relative to younger rats throughout the healing phase. Wound nerves were predominantly immunoreactive to calcitonin gene-related peptide, and synaptophysin-immunostaining revealed close associations between varicosities and myofibroblasts. These findings suggest that wound myofibroblasts may be a target of peripheral nerves, and delayed wound closure in mature rats is associated with deficiencies in both myofibroblasts and innervation.     

Establishment of hepatic and neural differentiation platforms of Wilson’s disease specific induced pluripotent stem cells

The combination of disease-specific human induced pluripotent stem cells (iPSC) and directed cell differentiation offers an ideal platform for modeling and studying many inherited human diseases. Wilson’s disease (WD) is a monogenic disorder of toxic copper accumulation caused by pathologic mutations of the ATP7B gene. WD affects multiple organs with primary manifestations in the liver and central nervous system (CNS). In order to better investigate the cellular pathogenesis of WD and to develop novel therapies against various WD syndromes, we sought to establish a comprehensive platform to differentiate WD patient iPSC into both hepatic and neural lineages. Here we report the generation of patient iPSC bearing a Caucasian population hotspot mutation of ATP7B. Combining with directed cell differentiation strategies, we successfully differentiated WD iPSC into hepatocyte-like cells, neural stem cells and neurons. Gene expression analysis and cDNA sequencing confirmed the expression of the mutant ATP7B gene in all differentiated cells. Hence we established a platform for studying both hepatic and neural abnormalities of WD, which may provide a new tool for tissue-specific disease modeling and drug screening in the future.     

Acutely reduced locomotor activity is a major contributor to Western diet-induced obesity in mice

The aim of the present study was to investigate the short- and long-term effects of a high-fat Western diet (WD) on intake, storage, expenditure, and fecal loss of energy as well as effects on locomotor activity and thermogenesis. WD for only 24 h resulted in a marked physiological shift in energy homeostasis, including increased body weight gain, body fat, and energy expenditure (EE) but an acutely lowered locomotor activity. The acute reduction in locomotor activity was observed after only 3-5 h on WD. The energy intake and energy absorption were increased during the first 24 h, lower after 72 h, and normalized between 7 and 14 days on WD compared with mice given chow diet. Core body temperature and EE was increased between 48 and 72 h but normalized after 21 days on WD. These changes paralleled plasma T(3) levels and uncoupling protein-1 expression in brown adipose tissue. After 21 days of WD, energy intake and absorption, EE, and body temperature were normalized. In contrast, the locomotor activity was reduced and body weight gain was increased over the entire 21-day study period on WD. Calculations based on the correlation between locomotor activity and EE in 2-h intervals at days 21-23 indicated that a large portion of the higher body weight gain in the WD group could be attributed to the reduced locomotor activity. In summary, an acute and persisting decrease in locomotor activity is most important for the effect of WD on body weight gain and obesity in mice.     

High Fat High Cholesterol Diet (Western Diet) Aggravates Atherosclerosis,Hyperglycemia and Renal Failure in Nephrectomized LDL Receptor Knockout Mice: Role of Intestine Derived Lipopolysaccharide

A high fat meal, frequently known as western diet (WD), exacerbates atherosclerosis and diabetes. Both these diseases are frequently associated with renal failure. Recent studies have shown that lipopolysaccharide (LPS) leaks into the circulation from the intestine in the setting of renal failure and after WD. However, it is not clear how renal function and associated disorders are affected by LPS. This study demonstrates that circulatory LPS exacerbates renal insufficiency, atherosclerosis and glucose intolerance. Renal insufficiency was induced by 2/3 nephrectomy in LDL receptor knockout mice. Nx animals were given normal diet (Nx) or WD (Nx+WD). The controls were sham operated animals on normal diet (control) and WD (WD). To verify if LPS plays a role in exaggerating renal insufficiency, polymyxin (PM), a known LPS antagonist, and curcumin (CU), a compound known to ameliorate chronic kidney disease (CKD), was given to Nx animals on western diet (Nx+WD+PM and Nx+WD+CU, respectively). Compared to control, all other groups displayed increased circulatory LPS. The Nx+WD cohort had the highest levels of LPS. Nx group had significant renal insufficiency and glucose intolerance but not atherosclerosis. WD had intense atherosclerosis and glucose intolerance but it did not show signs of renal insufficiency. Compared to other groups, Nx+WD had significantly higher cytokine expression, macrophage infiltration in the kidney, renal insufficiency, glucose intolerance and atherosclerosis. PM treatment blunted the expression of cytokines, deterioration of renal function and associated disorders, albeit not to the levels of Nx, and was significantly inferior to CU. PM is a non-absorbable antibiotic with LPS binding properties, hence its beneficial effect can only be due to its effect within the GI tract. We conclude that LPS may not cause renal insufficiency but can exaggerate kidney failure and associated disorders following renal insufficiency.     

A novel approach to oral apoA-I mimetic therapy

Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide, 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western diet (WD) at 2.2% by weight, and fed to LDL receptor-null (LDLR^−/−) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the aorta with lesions was 4.1 ± 4%, 3.3 ± 2.4%, and 1.9 ± 1.4% for WD, WD + EV, and WD + 6F, respectively (WD + 6F vs. WD, P = 0.0134; WD + 6F vs. WD + EV, P = 0.0386; WD + EV vs. WD, not significant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD + 6F mice; P < 0.0295. HDL cholesterol and paroxonase-1 activity (PON) were higher in WD + 6F mice (P = 0.0055 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (HETE) levels positively correlated with lesions (P < 0.0001); HDL cholesterol and PON were inversely correlated (P < 0.0001). After feeding WD + 6F: i) intact 6F was detected in small intestine (but not in plasma); ii) small intestine LPA was decreased compared with WD + EV (P < 0.0469); and iii) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions (P < 0.0179). These data suggest that 6F acts in the small intestine and provides a novel approach to oral apoA-I mimetic therapy.     

WD repeat domains target dictyostelium myosin heavy chain kinases by binding directly to myosin filaments

Myosin heavy chain kinase (MHCK) A phosphorylates mapped sites at the C-terminal tail of Dictyostelium myosin II heavy chain, driving disassembly of myosin filaments both in vitro and in vivo. MHCK A is organized into three functional domains that include an N-terminal coiled-coil region, a central kinase catalytic domain unrelated to conventional protein kinases, and a WD repeat domain at the C terminus. MHCK B is a homologue of MHCK A that possesses structurally related catalytic and WD repeat domains. In the current study, we explored the role of the WD repeat domains in defining the activities of both MHCK A and MHCK B using recombinant bacterially expressed truncations of these kinases either with or without their WD repeat domains. We demonstrate that substrate targeting is a conserved function of the WD repeat domains of both MHCK A and MHCK B and that this targeting is specific for Dictyostelium myosin II filaments. We also show that the mechanism of targeting involves direct binding of the WD repeat domains to the myosin substrate. To our knowledge, this is the first report of WD repeat domains physically targeting attached kinase domains to their substrates. The examples presented here may serve as a paradigm for enzyme targeting in other systems.     

Tree growth traits and social status affect the wood density of pioneer species in secondary subtropical forest

Wood density (WD) is not only an important parameter to estimate aboveground biomass but also an indicator of timber quality and plant adaptation strategies to stressful conditions (i.e., windthrow, pests, and pathogens). This study had three objectives: (1) to compare WD among seven subtropical tree species; (2) to determine how tree growth traits may influence possible differences in WD between the pioneer and shade‐tolerant species; and (3) to examine whether or not WD differs by tree social status (dominant vs. suppressed trees) within species. To do this, 70 trees were destructively harvested. From each tree, disks at different stem heights were obtained and subjected to a method of stem analysis to measure whole tree level WD. The results showed that WD differed significantly among the seven species (p < .001). Their average WD was 0.537 g/cm³, ranging from 0.409 g/cm³ for Choerospondias axillaris to 0.691 g/cm³ for Cyclobalanopsis glauca. The average WD of the four pioneer species (0.497 ± 0.13 g/cm³) was significantly lower (p < .01) than that of the three shade‐tolerant species (0.589 ± 0.12 g/cm³). The WD of the pioneers had a significant positive correlation with their stem diameter at breast height (DBH), tree height (H), and tree age, but WD had a significant negative correlation with relative growth rate (RGR). In contrast, the WD of the shade‐tolerant tree species had no significant relationships with DBH, H, tree age, or RGR. The dominant trees of the pioneer species had a higher WD than the suppressed trees, whereas the shade‐tolerant species had a lower WD for dominant trees than the suppressed trees. However, the differences in WD between dominant and suppressed trees were not significant. Taken together, the results suggest that classifying species into pioneer and shade‐tolerant groups to examine the effects of tree growth traits and social status could improve our understanding of intra‐ and interspecific variation in WD among subtropical tree species.     

WD-重复蛋白

 WD基元又称Trp-ASP或WD40,由40个左右的氨基酸残基组成,具有保守的GH和WD序列.WD基元存在于很多具有调控功能的蛋白质中,介导蛋白质之间的相互作用,在信号转导、蛋白运输、染色体修饰、转录或RNA加工等过程中具有重要作用.WD重复蛋白(WD-repeat protein)是含有多个保守的WD基元的蛋白质.近年发现,WD-repeat基因突变与人的几种疾病相关.本文对真核生物WD-重复蛋白的研究进展进行了综述,阐明了WD 重复蛋白的β-propeller结构特征及其作用机制,并对WD-重复蛋白的未来研究方向进行展望.     

Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson''s disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.     

Effects of short-term Western diet on cerebral oxidative stress and diabetes related factors in APP × PS1 knock-in mice

A chronic high fat Western diet (WD) promotes a variety of morbidity factors although experimental evidence for short-term WD mediating brain dysfunction remains to be elucidated. The amyloid precursor protein and presenilin-1 (APP × PS1) knock-in mouse model has been demonstrated to recapitulate some key features of Alzheimer's disease pathology, including amyloid-β (Aβ) pathogenesis. In this study, we placed 1-month-old APP × PS1 mice and non-transgenic littermates on a WD for 4 weeks. The WD resulted in a significant elevation in protein oxidation and lipid peroxidation in the brain of APP × PS1 mice relative to non-transgenic littermates, which occurred in the absence of increased Aβ levels. Altered adipokine levels were also observed in APP × PS1 mice placed on a short-term WD, relative to non-transgenic littermates. Taken together, these data indicate that short-term WD is sufficient to selectively promote cerebral oxidative stress and metabolic disturbances in APP × PS1 knock-in mice, with increased oxidative stress preceding alterations in Aβ. These data have important implications for understanding how WD may potentially contribute to brain dysfunction and the development of neurodegenerative disorders such as Alzheimer's disease.     

Xanthine oxidase inhibitor tungsten prevents the development of atherosclerosis in ApoE knockout mice fed a Western-type diet

Hyperlipidemia enhances xanthine oxidase (XO) activity. XO is an important source of reactive oxygen species (ROS). Since ROS are thought to promote atherosclerosis, we hypothesized that XO is involved in the development of atherosclerosis. ApoE(-/-) mice were fed a Western-type (WD) or control diet. In subgroups, tungsten (700 mg/L) was administered to inhibit XO. XO is a secreted enzyme which is formed in the liver as xanthine dehydrogenase (XDH) and binds to the vascular endothelium. High expression of XDH was found in the liver and WD increased liver XDH mRNA and XDH protein expression. WD induced the conversion of XDH to the radical-forming XO. Moreover, WD increased the hepatic expression of CD40, demonstrating activation of hepatic cells. Aortic tissue of ApoE(-/-) mice fed a WD for 6 months exhibited marked atherosclerosis, attenuated endothelium-dependent relaxation to acetylcholine, increased vascular oxidative stress, and mRNA expression of the chemokine KC. Tungsten treatment had no effect on plasma lipids but lowered the plasma XO activity. In animals fed a control diet, tungsten had no effect on radical formation, endothelial function, or atherosclerosis development. In mice fed a WD, however tungsten attenuated the vascular superoxide anion formation, prevented endothelial dysfunction, and attenuated KC mRNA expression. Most importantly, tungsten treatment largely prevented the development of atherosclerosis in the aorta of ApoE(-/-) mice on WD. Therefore, tungsten, potentially via the inhibition of XO, prevents the development of endothelial dysfunction and atherosclerosis in ApoE(-/-) mice on WD.     

Molecular analysis of Wilson patients: direct sequencing and MLPA analysis in the ATP7B gene and Atox1 and COMMD1 gene analysis

ATP7B mutations result in Cu storage in the liver and brain in Wilson disease (WD). Atox1 and COMMD1 were found to interact with ATP7B and involved in copper transport in the hepatocyte. To understand the molecular etiology of WD, we analyzed ATP7B, Atox1 and COMMD1 genes. Direct sequencing of (i) ATP7B gene was performed in 112 WD patients to identify the spectrum of disease-causing mutations in the French population, (ii) Atox1 gene was performed to study the known polymorphism 5'UTR-99T>C in 78 WD patients with two ATP7B mutations and (iii) COMMD1 gene was performed to detect the nucleotide change c.492GAT>GAC. MLPA (Multiplex Ligation-dependent Probe Amplification) analysis was performed in WD patients presenting only one ATP7B mutation. Among our 112 WD unrelated patients, 83 different ATP7B gene mutations were identified, 27 of which were novel. Two ATP7B mutations were identified in 98 WD cases, and one mutation was identified in 14 cases. In two of these 14 WD patients, we identified the deletion of exon 4 of the ATP7B gene by MLPA technique. In 78 selected patients of the cohort with two mutations in ATP7B, we have examined genotype-phenotype correlation between the detected changes in Atox1 and COMMD1 genes, and the presentation of the WD patients. Based on the data of this study, no major role can be attributed to Atox1 and COMMD in the pathophysiology or clinical variation of WD.     

Response of barley seedlings to water deficit and enhanced UV-B irradiation acting alone and in combination

Responses of barley seedlings to water deficit (WD) induced by polyethylene glycol (PEG 6000) and ultraviolet (UV-B; 280–320 nm) radiation and their interaction (UV-B + WD) were examined. A decrease in dry matter yield and water content of leaves and roots was observed following application of WD and UV-B + WD, while no changes were found after treating barley plants with UV-B. Proline content was increased in leaves under WD conditions and UV-B + WD. In contrast, UV-B treatment had no effect on the accumulation of proline in leaves of barley plants. Changes in root proline content showed a varied response: WD induced an increase in the level of this amino acid, while UV-B as well as UV-B + WD suppressed root proline content. The lipid peroxidation product malondialdehyde (MDA) was increased in leaves under WD and UV-B + WD stresses. Root MDA content increased in WD-stressed plants, but it decreased in the case of combined application of both stresses. The applied stress factors operated in a variable manner on phenylpropanoid metabolism. Phenylalanine ammonia-lyase (PAL) activity in leaves and roots was stimulated after exposure to WD and application of UV-B + WD stresses, while UV-B stress did not affect its activity. On the other hand, UV-B treatment enhanced the activity of 4:coumarate-CoA ligase (4CL) in leaves and this enhancement was positively correlated with the accumulation of anthocyanins and flavonols. However, the combined application of WD and UV-B reduced the positive effect of UV-B on the accumulation of these compounds and the activity of 4CL. Surprisingly, anthocyanins and flavonols were not detected in roots of examined barley seedlings despite increased 4CL activity. The results suggest that UV-B-induced activation of 4CL as well as accumulation of anthocyanin and flavonols in leaves is beneficial for the response to this stress factor. On the other hand, WD-induced reduction of the effect of UV-B on 4CL activity and the contents of anthocyanin and flavonol might be a cause of membrane damage in UV-B- and WD-stressed plants. In addition, conversely to what could be expected, the UV-B effect was perceived by the water-stressed roots, which exhibited reduced lipid peroxidation (MDA) and proline accumulation in WD-stressed plants exposed to UV-B.     

Proteomic analysis of the hepatic tissue of Long-Evans Cinnamon (LEC) rats according to the natural course of Wilson disease

Copper-induced toxicity is important in the pathogenic process of Wilson's disease (WD). Using Long-Evans Cinnamon (LEC) rats, an animal model of WD, the study was undertaken to identify proteins involved in the process of WD and to investigate their functional roles in copper-induced hepatotoxicity. In early stages, expression levels of mitochondrial matrix proteins including agmatinase, isovaleryl coenzyme A dehydrogenase, and cytochrome b5 were downregulated. As mitochondrial injuries progressed, along with subsequent apoptotic processes, expressions of malate dehydrogenase 1, annexin A5, transferrin, S-adenosylhomocysteine hydrolase, and sulfite oxidase 1 were differentially regulated. Notably, the expression of malate dehydrogenase 1 was downregulated while the annexin A5 was overexpressed in an age-dependent manner, indicating that these proteins may be involved in the WD process. In addition, pronounced under-expression of S-adenosylhomocysteine hydrolase in elderly LEC rats, also involved in monoamine neurotransmitter metabolism, indicates that this protein might be related to the development of neurological manifestations in WD. The results of our study help to understand the pathogenic process of WD in hepatic tissues, identifying the important proteins associated with the disease process of WD, and to investigate the molecular pathogenic process underlying the development of neurological manifestations in WD.     

The N-terminus of the WD5 repeat of human RACK1 binds to airway epithelial NHERF1

Regulation of the CFTR Cl channel function involves a protein complex of activated protein kinase Cepsilon (PKCepsilon) bound to RACK1, a receptor for activated C kinase, and RACK1 bound to the human Na(+)/H(+) exchanger regulatory factor (NHERF1) in human airway epithelial cells. Binding of NHERF1 to RACK1 is mediated via a NHERF1-PDZ1 domain. The goal of this study was to identify the binding motif for human NHERF1 on RACK1. We examined the site of binding of NHERF1 on RACK1 using peptides encoding the seven WD40 repeat units of human RACK1. One WD repeat peptide, WD5, directly binds NHERF1 and the PDZ1 domain with similar EC(50) values, blocks binding of recombinant RACK1 and NHERF1, and pulls down endogenous RACK1 from Calu-3 cell lysate in a dose-dependent manner. The remaining WD repeat peptides did not block RACK1-NHERF1 binding. An 11-amino acid peptide encoding a site on the PDZ1 domain blocks binding of the WD5 repeat peptide with the PDZ1 domain. An N-terminal 12-amino acid segment of the WD5 repeat peptide, which comprises the first of four antiparallel beta-strands, dose-dependently binds to the PDZ1 domain of NHERF1 and blocks binding of the PDZ1 domain to RACK1. These results suggest that the binding site might form a beta-turn with topology sufficient for binding of NHERF1. Our results also demonstrate binding of NHERF to RACK1 at the WD5 repeat, which is distinct from the PKCepsilon binding site on the WD6 repeat of RACK1.