PDZK1 and NHERF1 Regulate the Function of Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) by Modulating Its Subcellular Trafficking and Stability

The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (V_max: 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)⁻¹ in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability.     

Role of PDZK1 protein in apical membrane expression of renal sodium-coupled phosphate transporters

The sodium-dependent phosphate (Na/P(i)) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of P(i). The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in P(i) reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/P(i) transporters. Pdzk1(-/-) mice adapted to chronic low P(i) diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/P(i) transporters with NHERF-1 and PDZK1 by FRET. FRET measurements showed a much stronger interaction of NHERF-1 with NaPi-2a than with NaPi-2c. However, both Na/P(i) transporters showed similar FRET efficiencies with PDZK1. Interestingly, in cells adapted to low P(i) concentrations, there were increases in NaPi-2c/PDZK1 and NaPi-2a/NHERF-1 interactions. The differential affinity of the Na/P(i) transporters for NHERF-1 and PDZK1 proteins could partially explain their differential regulation and/or stability in the apical membrane. In this regard, direct interaction between NaPi-2c and PDZK1 seems to play an important role in the physiological regulation of NaPi-2c.     

NHERF2/NHERF3 Protein Heterodimerization and Macrocomplex Formation Are Required for the Inhibition of NHE3 Activity by Carbachol

NHERF1, NHERF2, and NHERF3 belong to the NHERF (Na⁺/H⁺ exchanger regulatory factor) family of PSD-95/Discs-large/ZO-1 (PDZ) scaffolding proteins. Individually, each NHERF protein has been shown to be involved in the regulation of multiple receptors or transporters including Na⁺/H⁺ exchanger 3 (NHE3). Although NHERF dimerizations have been reported, results have been inconsistent, and the physiological function of NHERF dimerizations is still unknown. The current study semiquantitatively compared the interaction strength among all possible homodimerizations and heterodimerizations of these three NHERF proteins by pulldown and co-immunoprecipitation assays. Both methods showed that NHERF2 and NHERF3 heterodimerize as the strongest interaction among all NHERF dimerizations. In vivo NHERF2/NHERF3 heterodimerization was confirmed by FRET and FRAP (fluorescence recovery after photobleach). NHERF2/NHERF3 heterodimerization is mediated by PDZ domains of NHERF2 and the C-terminal PDZ domain recognition motif of NHERF3. The NHERF3-4A mutant is defective in heterodimerization with NHERF2 and does not support the inhibition of NHE3 by carbachol. This suggests a role for NHERF2/NHERF3 heterodimerization in the regulation of NHE3 activity. In addition, both PDZ domains of NHERF2 could be simultaneously occupied by NHERF3 and another ligand such as NHE3, α-actinin-4, and PKCα, promoting formation of NHE3 macrocomplexes. This study suggests that NHERF2/NHERF3 heterodimerization mediates the formation of NHE3 macrocomplexes, which are required for the inhibition of NHE3 activity by carbachol.     

NHERF3 (PDZK1) Contributes to Basal and Calcium Inhibition of NHE3 Activity in Caco-2BBe Cells

Elevated intracellular Ca²⁺ ([Ca²⁺]_i) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca²⁺]_i regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca²⁺ ionophore, 4-bromo-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 (0.5 μm): 1) inhibited NHE3 V_max activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca²⁺]_i by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca²⁺]_i inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca²⁺]_i; 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca²⁺.In normal digestive physiology, the brush border (BB)² Na⁺/H⁺ exchanger, NHE3, mediates the majority of the NaCl and NaHCO₃ absorption in the ileum (1). Sequential inhibition and stimulation of NHE3 occur as part of digestive physiology. Short-term regulation of NHE3 activity is achieved through a variety of factors that affect NHE3 turnover number and/or surface expression and often involve a role for the cytoskeleton and accessory proteins, including the multi-PDZ domain containing proteins, NHERF1 and NHERF2 (1, 2). However, many details of this regulation are not understood.The NHERF (Na⁺/H⁺ exchanger regulatory factor) family of multi-PDZ domain containing proteins consists of four evolutionarily related members, all of which are expressed in epithelial cells of the mammalian small intestine (2). NHERF1 and NHERF2 have been previously shown to contribute to acute NHE3 stimulation and inhibition (3–10). Recently, two additional PDZ domain containing proteins, termed NHERF3/PDZK1 and NHERF4/PDZK2/IKEPP, have been demonstrated to possess sequence homology with NHERF1 and NHERF2 (11–14). However, unlike NHERF1 and NHERF2, which are comprised of two tandem PDZ domains flanked by a C-terminal ezrin/radixin/moesin binding domain, NHERF3 and NHERF4 consist of four PDZ domains but no other protein-protein interacting domains (12).NHERF3 was initially identified by a yeast two-hybrid screen from a human kidney cDNA library using the membrane-associated protein MAP17, as bait (12). NHERF3 is expressed in the brush border of epithelial cells of the kidney proximal tubule and the small intestine (12). NHERF3 associates with and, in a few cases, has been shown to regulate the activity of multiple apical membrane ion transporters including the cystic fibrosis transmembrane regulator (CFTR), urate anion exchanger 1 (URAT1), sodium-phosphate cotransporter type IIa (NaP_iIIa), proton-coupled peptide transporter (PEPT2), and organic cation/carnitine cotransporter (OCTN2) (15–19). Furthermore, NHERF3 directly binds the C terminus of NHE3 (20). Recent studies have begun evaluating the effect of NHERF3 on mouse intestinal Na⁺ and Cl⁻ transport. Basal electroneutral sodium absorption was decreased by >40% in the NHERF3 null mouse jejunum (21) and by >80% in the colon (22). In addition, Cinar et al. (22) demonstrated that cAMP and [Ca²⁺]_i inhibition of NHE3 activity was abolished in the NHERF3 null mouse colon. However, the mechanism by which NHERF3 regulates NHE3 activity was not resolved.Several physiological and pathophysiological agonists, acting through [Ca²⁺]_i-induced second messenger systems, are known to inhibit electroneutral NaCl absorption in the small intestine (1, 23). Elevation of [Ca²⁺]_i has previously been demonstrated to inhibit NHE3 activity in a NHERF2-, but not NHERF1-dependent manner (5). NHERF2 regulation of NHE3 involves the formation of multiprotein complexes at the plasma membrane that include NHE3, NHERF2, α-actinin-4, and PKCα, which induce endocytic removal of NHE3 from the plasma membrane by a PKC-dependent mechanism (5, 24). Because multiple PDZ proteins exist in the apical pole of epithelial cells (2), the current study was designed to determine whether NHERF3 could reconstitute Ca²⁺ regulation of NHE3 activity and to define how that occurred.     

The characterization of myosin–product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction

Evidence is presented that the myosin subfragment-1–ADP complex, generated by the addition of Mg²⁺ and ADP to subfragment 1, is an intermediate within the myosin Mg²⁺-dependent adenosine triphosphatase (ATPase) turnover cycle. The existence of this species as a steady-state intermediate at pH8 and 5°C is demonstrated by fluorescence measurements, but its concentration becomes too low to measure at 21°C. This arises because there is a marked temperature-dependence on the rate of the process controlling ADP dissociation from subfragment 1 (rate=1.4s⁻¹ at 21°C, 0.07s⁻¹ at 5°C). In the ATPase pathway this reaction is in series with a relatively temperature-insensitive process, namely an isomerization of the subfragment-1–product complex (rate=0.055s⁻¹ at 21°C, 0.036s⁻¹ at 5°C). By means of studies on the P_i inhibition of nucleotide-association rates, a myosin subfragment-1–P_i complex was characterized with a dissociation equilibrium constant of 1.5mm. P_i appears to bind more weakly to the myosin subfragment-1–ADP complex. The studies indicate that P_i dissociates from subfragment 1 at a rate greater than 40s⁻¹, and substantiates the existence of a myosin-product isomerization before product release in the elementary processes of the Mg²⁺-dependent ATPase. In this ATPase mechanism Mg²⁺ associates as a complex with ATP and is released as a complex with ADP. In 0.1m-KCl at pH8 1.0mol of H⁺ is released/mol of subfragment 1 concomitant with the myosin-product isomerization or P_i dissociation, and 0.23 mol of H⁺ is released/mol of subfragment when ATP binds to the protein, but 0.23 mol of H⁺ is taken up again from the medium when ADP dissociates. Within experimental sensitivity no H⁺ is released into the medium in the step involving ATP cleavage.     

Chloride Conductance and P i Transport are Separate Functions Induced by the Expression of NaPi-1 in Xenopus Oocytes

Expression of the protein NaPi-1 in Xenopus oocytes has previously been shown to induce an outwardly rectifying Cl⁻ conductance (G_Cl), organic anion transport and Na⁺-dependent P _i -uptake. In the present study we investigated the relation between the NaPi-1 induced G_Cl and P _i -induced currents and transport. NaPi-1 expression induced P _i -transport, which was not different at 1–20 ng/oocyte NaPi-1 cRNA injection and was already maximal at 1–2 days after cRNA injection. In contrast, G_Cl was augmented at increased amounts of cRNA injection (1–20 ng/oocyte) and over a five day expression period. Subsequently all experiments were performed on oocytes injected with 20 ng/oocytes cRNA. P _i -induced currents (Ip) could be observed in NaPi-1 expressing oocytes at high concentrations of P _i (≥ 1 mm P _i ). The amplitudes of Ip correlated well with G_Cl. Ip was blocked by the Cl⁻ channel blocker NPPB, partially Na⁺-dependent and completely abolished in Cl⁻ free solution. In contrast, P _i -transport in NaPi-1 expressing oocytes was not NPPB sensitive, stronger depending on extracellular Na⁺ and weakly affected by Cl⁻ substitution. Endogenous P _i -uptake in water-injected oocytes amounted in all experiments to 30–50% of the Na⁺-dependent P _i -transport observed in NaPi-1 expressing oocytes. The properties of the endogenous P _i -uptake system (K _m for P _i > 1 mm; partial Na⁺- and Cl⁻-dependence; lack of NPPB block) were similar to the NaPi-1 induced P _i -uptake, but no Ip could be recorded at P _i -concentrations ≤3 mm. In summary, the present data suggest that Ip does not reflect charge transfer related to P _i -uptake, but a P _i -mediated modulation of G_Cl. Received: 22 October 1997/Revised: 24 March 1998     

Na/H Exchanger Regulatory Factors Control Parathyroid Hormone Receptor Signaling by Facilitating Differential Activation of Gα Protein Subunits

The Na/H exchanger regulatory factors, NHERF1 and NHERF2, are adapter proteins involved in targeting and assembly of protein complexes. The parathyroid hormone receptor (PTHR) interacts with both NHERF1 and NHERF2. The NHERF proteins toggle PTHR signaling from predominantly activation of adenylyl cyclase in the absence of NHERF to principally stimulation of phospholipase C when the NHERF proteins are expressed. We hypothesized that this signaling switch occurs at the level of the G protein. We measured G protein activation by [³⁵S]GTPγS binding and Gα subtype-specific immunoprecipitation using three different cellular models of PTHR signaling. These studies revealed that PTHR interactions with NHERF1 enhance receptor-mediated stimulation of Gα_q but have no effect on stimulation of Gα_i or Gα_s. In contrast, PTHR associations with NHERF2 enhance receptor-mediated stimulation of both Gα_q and Gα_i but decrease stimulation of Gα_s. Consistent with these functional data, NHERF2 formed cellular complexes with both Gα_q and Gα_i, whereas NHERF1 was found to interact only with Gα_q. These findings demonstrate that NHERF interactions regulate PTHR signaling at the level of G proteins and that NHERF1 and NHERF2 exhibit isotype-specific effects on G protein activation.     

Nuclear NHERF1 expression as a prognostic marker in breast cancer

Our purpose was to investigate whether Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) expression could be linked to prognosis in invasive breast carcinomas. NHERF1, an ezrin-radixin-moesin (ERM) binding phosphoprotein 50, is involved in the linkage of integral membrane proteins to the cytoskeleton. It is therefore believed to have an important role in cell signaling associated with changes in cell cytoarchitecture. NHERF1 expression is observed in various types of cancer and is related to tumor aggressiveness. To date the most extensive analyses of the influence of NHERF1 in cancer development have been performed on breast cancer. However, the underlying mechanism and its prognostic significance are still undefined. NHERF1 expression was studied by immunohistochemistry (IHC) in a cohort of 222 breast carcinoma patients. Association of cytoplasmic and nuclear NHERF1 expression with survival was analyzed. Disease-free survival (DFS) and overall survival (OS) were determined based on the Kaplan–Meier method. Cytoplasmic NHERF1 expression was associated with negative progesterone receptor (PgR) (P=0.017) and positive HER2 expression (P=0.023). NHERF1 also showed a nuclear localization and this correlated with small tumor size (P=0.026) and positive estrogen receptor (ER) expression (P=0.010). Multivariate analysis identified large tumor size (P=0.011) and nuclear NHERF1 expression (P=0.049) to be independent prognostic variables for DFS. Moreover, the nuclear NHERF1(−)/ER(−) immunophenotype (27%) was statistically associated with large tumor size (P=0.0276), high histological grade (P=0.0411), PgR-negative tumors (P<0.0001) and high proliferative activity (P=0.0027). These patients had worse DFS compared with patients with nuclear NHERF1(+)/ER(+) tumors (75.4% versus 92.6% P=0.010). These results show that the loss of nuclear NHERF1 expression is associated with reduced survival, and the link between nuclear NHERF1 and ER expression may serve as a prognostic marker for the routine clinical management of breast cancer patients.     

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.     

Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

The Na/H exchanger 3 (NHE3) and the Cl/HCO₃ exchanger down-regulated in adenoma (DRA) together facilitate intestinal electroneutral NaCl absorption. Elevated Ca²⁺_i inhibits NHE3 through mechanisms involving the PDZ domain proteins NHE3 kinase A regulatory protein (E3KARP) or PDZ kidney 1 (PDZK1). DRA also possesses a PDZ-binding motif, but the roles of interactions with E3KARP or PDZK1 and Ca²⁺_i in DRA regulation are unknown. Wild type DRA and a mutant lacking the PDZ interaction motif (DRA-ETKFminus) were expressed constitutively in human embryonic kidney (HEK) and inducibly in Caco-2/BBE cells. DRA-mediated Cl/HCO₃ exchange was measured as intracellular pH changes. Ca²⁺_i was assessed fluorometrically. DRA was induced 8–16-fold and was delivered to the apical surface of polarized Caco-2 cells. Putative anion transporter 1 and cystic fibrosis transmembrane regulator did not contribute to Cl/HCO₃ exchange in transfected Caco-2 cells. The calcium ionophore 4Br-{"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 inhibited DRA and DRA-ETKFminus in HEK cells, but only full-length DRA was inhibited in Caco-2 cells. In contrast, 100 μm UTP, which increased Ca²⁺_i, inhibited full-length DRA but not DRA-ETKFminus in Caco-2 and HEK cells. In HEK cells, which express little PDZK1, additional transfection of PDZK1 was required for UTP to inhibit DRA. As HEK cells do not express cystic fibrosis transmembrane regulator or NHE3, the data indicate that Ca²⁺_i-dependent DRA inhibition is not because of modulation of other transport activities. In polarized epithelium, this inhibition requires interaction of DRA with PDZK1. Together with data from PDZK1^−/− mice, these data underscore the prominent role of PDZK1 in Ca²⁺_i-mediated inhibition of colonic NaCl absorption.In the gastrointestinal tract electroneutral NaCl absorption occurs in the distal ileum and proximal colon by parallel Na/H exchange and Cl/HCO₃ exchange. Studies in knock-out mice have confirmed that NHE3² (Na/H exchanger, isoform 3; SLC9A3) and DRA (down-regulated in adenoma; SLC26A3) are the primary transporters responsible for these events (1, 2). The importance of the latter is emphasized by the human genetic disorder congenital chloride diarrhea (3), in which a nonfunctional DRA leads to life-threatening diarrhea. DRA is also expressed in the duodenum (in the lower villus) and in the pancreas (4–6), where it is involved in chloride and bicarbonate secretion together with CFTR (4–7). All three transport proteins, NHE3, DRA, and CFTR, have PDZ interaction motifs that facilitate binding to several members of the NHERF class of PDZ adapter proteins (8, 9).Electroneutral NaCl absorption is regulated largely in parallel but reciprocally with electrogenic chloride secretion (10). In different systems NHE3 is acutely regulated by cAMP, cGMP, intracellular calcium, lysophosphatidic acid, and epidermal growth factor (11) as well as by tumor necrosis factor-α (12). Notably, some of these regulatory processes are mediated through the interaction of NHE3 with several members of the NHERF class of PDZ adapter proteins (8, 11).Relatively little is known about the acute regulation of DRA. In the context of chloride and bicarbonate secretion, DRA is activated by cAMP, if it is expressed in a complex with CFTR and PDZ adapter proteins (PDZK1, also known as CAP70, and/or NHERF) (6, 7, 13). It is expected that DRA is inhibited in vivo in parallel with NHE3 during NaCl absorption, and in Caco-2/BBE cells transfected with NHE3 and DRA, this coupled inhibition has recently been shown (14). In Xenopus oocytes DRA is refractory to regulation by the calmodulin antagonist calmidazolium (10 μm), the PP2A inhibitor calyculin A (100 nm), or actin-modifying agents (13). Other data suggest that direct phosphorylation may regulate DRA, as mutation of tyrosine 756 (Y756F) increases DRA activity, although no signaling pathway has been suggested (13). Thus the regulation of DRA remains poorly understood. Moreover, no data address whether DRA regulation can occur independently or is always dependent on regulation of partner transporters, i.e. CFTR or NHE3, to which it is functionally and structurally coupled.Here we show that DRA activity is inhibited by elevations of Ca²⁺_i, that this regulation is independent of CFTR and NHE3, and that regulation requires interactions between DRA and the PDZ adapter protein PDZK1.     

Structural insights into PDZ-mediated interaction of NHERF2 and LPA2, a cellular event implicated in CFTR channel regulation

The formation of CFTR–NHERF2–LPA₂ macromolecular complex in airway epithelia regulates CFTR channel function and plays an important role in compartmentalized cAMP signaling. We previously have shown that disruption of the PDZ-mediated NHERF2–LPA₂ interaction abolishes the LPA inhibitory effect and augments CFTR Cl⁻ channel activity in vitro and in vivo. Here we report the first crystal structure of the NHERF2 PDZ1 domain in complex with the C-terminal LPA₂ sequence. The structure reveals that the PDZ1–LPA₂ binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four LPA₂ residues contributing to specific interactions. Comparison of the PDZ1–LPA₂ structure to the structure of PDZ1 in complex with a different peptide provides insights into the diverse nature of PDZ1 substrate recognition and suggests that the conformational flexibility in the ligand binding pocket is involved in determining the broad substrate specificity of PDZ1. In addition, the structure reveals a small surface pocket adjacent to the ligand-binding site, which may have therapeutic implications. This study provides an understanding of the structural basis for the PDZ-mediated NHERF2–LPA₂ interaction that could prove valuable in selective drug design against CFTR-related human diseases.     

Ezrin Induces Long-Range Interdomain Allostery in the Scaffolding Protein NHERF1

Scaffolding proteins are molecular switches that control diverse signaling events. The scaffolding protein Na⁺/H⁺ exchanger regulatory factor 1 (NHERF1) assembles macromolecular signaling complexes and regulates the macromolecular assembly, localization, and intracellular trafficking of a number of membrane ion transport proteins, receptors, and adhesion/antiadhesion proteins. NHERF1 begins with two modular protein-protein interaction domains—PDZ1 and PDZ2—and ends with a C-terminal (CT) domain. This CT domain binds to ezrin, which, in turn, interacts with cytosekeletal actin. Remarkably, ezrin binding to NHERF1 increases the binding capabilities of both PDZ domains. Here, we use deuterium labeling and contrast variation neutron-scattering experiments to determine the conformational changes in NHERF1 when it forms a complex with ezrin. Upon binding to ezrin, NHERF1 undergoes significant conformational changes in the region linking PDZ2 and its CT ezrin-binding domain, as well as in the region linking PDZ1 and PDZ2, involving very long range interactions over 120 Å. The results provide a structural explanation, at mesoscopic scales, of the allosteric control of NHERF1 by ezrin as it assembles protein complexes. Because of the essential roles of NHERF1 and ezrin in intracellular trafficking in epithelial cells, we hypothesize that this long-range allosteric regulation of NHERF1 by ezrin enables the membrane-cytoskeleton to assemble protein complexes that control cross-talk and regulate the strength and duration of signaling.     

Noncanonical Role of the PDZ4 Domain of the Adaptor Protein PDZK1 in the Regulation of the Hepatic High Density Lipoprotein Receptor Scavenger Receptor Class B,Type I (SR-BI)

The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI''s cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1''s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.     

Gating Kinetics of Single Large-Conductance Ca2+-Activated K+ Channels in High Ca2+ Suggest a Two-Tiered Allosteric Gating Mechanism?

The Ca²⁺-dependent gating mechanism of large-conductance calcium-activated K⁺ (BK) channels from cultured rat skeletal muscle was examined from low (4 μM) to high (1,024 μM) intracellular concentrations of calcium (Ca²⁺ _i) using single-channel recording. Open probability (P _o) increased with increasing Ca²⁺ _i (K _0.5 11.2 ± 0.3 μM at +30 mV, Hill coefficient of 3.5 ± 0.3), reaching a maximum of ∼0.97 for Ca²⁺ _i ∼ 100 μM. Increasing Ca²⁺ _i further to 1,024 μM had little additional effect on either P _o or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca²⁺ _i (>100 μM), compared with three to four open and five to seven closed states at lower Ca²⁺ _i. The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of two-dimensional (2-D) dwell-time distributions obtained from low to high Ca²⁺ _i. Kinetic schemes drawn from the 10-state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca²⁺ _i. Kinetic schemes drawn from Eigen''s general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca²⁺ _i. However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca²⁺ _i. In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca²⁺ _i. A model that assumed that the apparent Ca²⁺-binding steps can reach a maximum rate at high Ca²⁺ _i could also approximate the gating from low to high Ca²⁺ _i. The considered models can serve as working hypotheses for the gating of BK channels.     

Enhanced eIF1 binding to the 40S ribosome impedes conformational rearrangements of the preinitiation complex and elevates initiation accuracy

In the current model of translation initiation by the scanning mechanism, eIF1 promotes an open conformation of the 40S subunit competent for rapidly loading the eIF2·GTP·Met-tRNA_i ternary complex (TC) in a metastable conformation (P_OUT) capable of sampling triplets entering the P site while blocking accommodation of Met-tRNA_i in the P_IN state and preventing completion of GTP hydrolysis (P_i release) by the TC. All of these functions should be reversed by eIF1 dissociation from the preinitiation complex (PIC) on AUG recognition. We tested this model by selecting eIF1 Ssu⁻ mutations that suppress the elevated UUG initiation and reduced rate of TC loading in vivo conferred by an eIF1 (Sui⁻) substitution that eliminates a direct contact of eIF1 with the 40S subunit. Importantly, several Ssu⁻ substitutions increase eIF1 affinity for 40S subunits in vitro, and the strongest-binding variant (D61G), predicted to eliminate ionic repulsion with 18S rRNA, both reduces the rate of eIF1 dissociation and destabilizes the P_IN state of TC binding in reconstituted PICs harboring Sui⁻ variants of eIF5 or eIF2. These findings establish that eIF1 dissociation from the 40S subunit is required for the P_IN mode of TC binding and AUG recognition and that increasing eIF1 affinity for the 40S subunit increases initiation accuracy in vivo. Our results further demonstrate that the GTPase-activating protein eIF5 and β-subunit of eIF2 promote accuracy by controlling eIF1 dissociation and the stability of TC binding to the PIC, beyond their roles in regulating GTP hydrolysis by eIF2.     

Formation of a Ternary Complex among NHERF1, β-Arrestin,and Parathyroid Hormone Receptor

β-Arrestins are crucial regulators of G-protein coupled receptor (GPCR) signaling, desensitization, and internalization. Despite the long-standing paradigm that agonist-promoted receptor phosphorylation is required for β-arrestin2 recruitment, emerging evidence suggests that phosphorylation-independent mechanisms play a role in β-arrestin2 recruitment by GPCRs. Several PDZ proteins are known to interact with GPCRs and serve as cytosolic adaptors to modulate receptor signaling and trafficking. Na⁺/H⁺ exchange regulatory factors (NHERFs) exert a major role in GPCR signaling. By combining imaging and biochemical and biophysical methods we investigated the interplay among NHERF1, β-arrestin2, and the parathyroid hormone receptor type 1 (PTHR). We show that NHERF1 and β-arrestin2 can independently bind to the PTHR and form a ternary complex in cultured human embryonic kidney cells and Chinese hamster ovary cells. Although NHERF1 interacts constitutively with the PTHR, β-arrestin2 binding is promoted by receptor activation. NHERF1 interacts directly with β-arrestin2 without using the PTHR as an interface. Fluorescence resonance energy transfer studies revealed that the kinetics of PTHR and β-arrestin2 interactions were modulated by NHERF1. These findings suggest a model in which NHERF1 may serve as an adaptor, bringing β-arrestin2 into close proximity to the PTHR, thereby facilitating β-arrestin2 recruitment after receptor activation.     

α-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine

The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and α-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether α-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with α-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to α-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with α-AP-2. siRNA silencing of α-AP-2 in cells significantly reduced CFTR endocytosis, further supporting α-AP-2 as the direct binding partner for CFTR. α-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snell''s Waltzer myosin VI^(sv/sv) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI^(sv/sv) enterocytes. The findings support α-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.     

Na+/H+ Exchanger Regulatory Factor 1 (NHERF1) Directly Regulates Osteogenesis

Bone formation requires synthesis, secretion, and mineralization of matrix. Deficiencies in these processes produce bone defects. The absence of the PDZ domain protein Na⁺/H⁺ exchange regulatory factor 1 (NHERF1) in mice, or its mutation in humans, causes osteomalacia believed to reflect renal phosphate wasting. We show that NHERF1 is expressed by mineralizing osteoblasts and organizes Na⁺/H⁺ exchangers (NHEs) and the PTH receptor. NHERF1-null mice display reduced bone formation and wide mineralizing fronts despite elimination of phosphate wasting by dietary supplementation. Bone mass was normal, reflecting coordinated reduction of bone resorption and formation. NHERF1-null bone had decreased strength, consistent with compromised matrix quality. Mesenchymal stem cells from NHERF1-null mice showed limited osteoblast differentiation but enhanced adipocyte differentiation. PTH signaling and Na⁺/H⁺ exchange were dysregulated in these cells. Osteoclast differentiation from monocytes was unaffected. Thus, NHERF1 is required for normal osteoblast differentiation and matrix synthesis. In its absence, compensatory mechanisms maintain bone mass, but bone strength is reduced.     

Characterization of Two Inducible Phosphate Transport Systems in Rhizobium tropici

Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P_i). To better understand phosphorus movement between the bacteroid and the host plant, P_i transport was characterized in R. tropici. We observed two P_i transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K_m and V_max values for the low-affinity system were estimated to be 34 ± 3 μM P_i and 118 ± 8 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively, and the K_m and V_max values for the high-affinity system were 0.45 ± 0.01 μM P_i and 86 ± 5 nmol of P_i · min⁻¹ · mg (dry weight) of cells⁻¹, respectively. Both systems were inducible by P_i starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P_i transport through both systems was eliminated by the ATPase inhibitor N,N′-dicyclohexylcarbodiimide; the P_i transport rate was correlated with the intracellular ATP concentration. Also, P_i movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P_i transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity.