Ezrin/radixin/moesin (ERM) proteins function as general cross-linkers between plasma membrane proteins and the actin cytoskeleton and are involved in the functional expression of membrane proteins on the cell surface. They also integrate Rho guanosine 5′-triphosphatase (GTPase) signaling to regulate cytoskeletal organization by sequestering Rho-related proteins. They act as protein kinase A (PKA)-anchoring proteins and sequester PKA close to its target proteins for their effective phosphorylation and functional regulation. Therefore, ERM proteins seem to play important roles in the membrane transport of electrolytes by ion channels and transporters. In this review, we focus on the pathophysiological roles of ERM proteins in in vivo studies and introduce the phenotypes of their knockout and knockdown mice.Key words ezrin/radixin/moesin protein; cytoskeleton; knockout mouse EZRIN/RADIXIN/MOESIN PROTEINS REGULATE CYTOSKELETAL ORGANIZATION BY CROSS-LINK-ING PLASMA MEMBRANES WITH THE ACTIN CY-TOSKELETON AND INTEGRATING RHO GTPASE SIGNALINGThe ezrin, radixin, and moesin (ERM) proteins are general cross-linkers between cortical actin filaments and plasma membranes. They are concentrated at cell surface structures such as microvilli, filopodia, uropods, ruffling membranes, retraction fibers, and cell adhesion sites where actin filaments are associated with plasma membranes, but not along cytoplasmic actin filaments such as stress fibers.1) ERM proteins also integrate Rho guanosine 5′-triphosphatase (GTPase) signaling to regulate cytoskeletal organization by sequestering Rhorelated proteins.2) The apparent molecular mass of ERM is 82, 80, and 75 kDa, respectively. They show high amino acid identity, especially in their amino-and carboxy-terminal domains. Their amino-terminal domains consisting of ca. 300 amino acid residues are termed "the band four point one and ERM (FERM) domain" because their amino acid sequences are conserved within ERM proteins and the erythrocyte band 4.1 protein (Fig. 1). FERM domains are also found in numerous membrane-associated signaling and cytoskeletal proteins such as talin.3) The FERM domains, which are composed of three structural modules (F1, F2, and F3), together form a compact clover-shaped structure 4,5) and bind to integral membrane proteins, [6][7][8][9][10][11][12][13][14][15][16] scaffold proteins, [17][18][19][20] and the Rho-related proteins (such as the Rho-guanosine 5′-diphosphate (GDP)-dissociation inhibitor [Rho-GDI] and Dbl) 21,22) listed in Table 1, as well as to phosphatidylinositol 4,5-bisphosphate (PIP 2 ). 12) The scaffold proteins Na + /H + exchanger regulatory factors (NHERF) 1 and 2, which contain two PDZ (PSD-95, Discs-large, and ZO-1) domains, bind to the FERM domain at the carboxy-terminus. 17,20) These PDZ domains on the NHERFs interact with the PDZ-binding motif of membrane proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Na 19,25,26) On the other hand, the carboxy-terminal domains, especially 34 amino acid residues, are also h...
We identified a novel prostaglandin (PG)-specific organic anion transporter (OAT) in the OAT group of the SLC22 family. The transporter designated OAT-PG from mouse kidney exhibited Na ؉ -independent and saturable transport of PGE 2 when expressed in a proximal tubule cell line (S 2 ). Unusual for OAT members, OAT-PG showed narrow substrate selectivity and high affinity for a specific subset of PGs, including PGE 2 , PGF 2␣ , and PGD 2 . Similar to PGE 2 receptor and PGT, a structurally distinct PG transporter, OAT-PG requires for its substrates an ␣-carboxyl group, with a double bond between C13 and C14 as well as a (S)-hydroxyl group at C15. Unlike the PGE 2 receptor, however, the hydroxyl group at C11 in a cyclopentane ring is not essential for OAT-PG substrates. Addition of a hydroxyl group at C19 or C20 impairs the interaction with OAT-PG, whereas an ethyl group at C20 enhances the interaction, suggesting the importance of hydrophobicity around the -tail tip forming a "hydrophobic core" accompanied by a negative charge, which is essential for substrates of OAT members. OAT-PG-mediated transport is concentrative in nature, although OAT-PG mediates both facilitative and exchange transport. OAT-PG is kidney-specific and localized on the basolateral membrane of proximal tubules where a PG-inactivating enzyme, 15-hydroxyprostaglandin dehydrogenase, is expressed. Because of the fact that 15-keto-PGE 2 , the metabolite of PGE 2 produced by 15-hydroxyprostaglandin dehydrogenase, is not a substrate of OAT-PG, the transport-metabolism coupling would make unidirectional PGE 2 transport more efficient. By removing extracellular PGE 2 , OAT-PG is proposed to be involved in the local PGE 2 clearance and metabolism for the inactivation of PG signals in the kidney cortex.
Ezrin cross-links plasma membrane proteins with the actin cytoskeleton. In the kidney, ezrin mainly localizes at the brush border membrane of proximal tubules with the scaffolding protein, Na(+)/H(+) exchanger regulatory factor (NHERF) 1. NHERF1 interacts with the sodium/phosphate cotransporter, Npt2a. Defects in NHERF1 or Npt2a in mice cause hypophosphatemia. Here we studied the physiological role of ezrin in renal phosphate reabsorption using ezrin knockdown mice (Vil2). These mice exhibit hypophosphatemia, hypocalcemia, and osteomalacia. The reduced plasma phosphate concentrations were ascribed to defects in urinary phosphate reabsorption. Immunofluorescence and immunoblotting indicated a marked reduction in renal Npt2a and NHERF1 expression at the apical membrane of proximal tubules in the knockdown mice. On the other hand, urinary loss of calcium was not found in Vil2 mice. Plasma concentrations of 1,25-dihydroxyvitamin D were elevated following reduced plasma phosphate levels, and mRNA of the vitamin D-dependent TRPV6 calcium channel were significantly increased in the duodenum of knockdown mice. Expression of TRPV6 at the apical membrane, however, was significantly decreased. Furthermore, tibial bone mineral density was significantly lower in both the adult and young Vil2 mice. These results suggest that ezrin is required for the regulation of systemic phosphate and calcium homeostasis in vivo.
Cholangiopathies share common features, including bile duct proliferation, periportal fibrosis, and intrahepatic cholestasis. Damage of biliary epithelium by autoimunne disorder, virus infection, toxic compounds, and developmental abnormalities causes severe progressive hepatic disorders responsible for high mortality. However, the etiologies of these cholestatic diseases remain unclear because useful models to study the pathogenic mechanisms are not available. In the present study, we have found that ezrin knockdown (Vil2kd/kd) mice develop severe intrahepatic cholestasis characterized by extensive bile duct proliferation, periductular fibrosis, and intrahepatic bile acid accumulation without developmental defects of bile duct morphology and infiltration of inflammatory cells. Ezrin is a membrane cytoskeletal cross-linker protein, which is known to interact with transporters, scaffold proteins, and actin cytoskeleton at the plasma membrane. We found that the normal apical membrane localizations of several transport proteins including cystic fibrosis transmembrane conductance regulator (CFTR), anion exchanger 2 (AE-2), aquaporin 1 (AQP1), and Na+/H+ exchanger regulatory factor were disturbed in bile ducts of Vil2kd/kd mice. Stable expression of a dominant negative form of ezrin in immortalized mouse cholangiocytes also led to the reduction of the surface expression of CFTR, AE-2, and AQP1. Reduced surface expression of these transport proteins was accompanied by reduced functional expression, as evidenced by the fact these cells exhibited decreased CFTR-mediated Cl− efflux activity. Furthermore, bile flow and biliary HCO3− concentration were also significantly reduced in Vil2kd/kd mice. Conclusion: Dysfunction of ezrin mimics important aspects of the pathological mechanisms responsible for cholangiopathies. The Vil2kd/kd mouse may be a useful model to exploit in the development and testing of potential therapies for cholangiopathies.
Based on the nucleotide sequence of a mouse prostaglandin-specific transporter (mOAT-PG), we identified a rat homolog (rOAT-PG) which shares 80% identity with mOAT-PG in a deduced amino acid sequence. rOAT-PG transports PGE(2) and colocalizes with 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a metabolic enzyme for PGs, in proximal tubules, suggesting that rOAT-PG is involved in PGE(2) clearance to regulate its physiological function in the renal cortex. We found that the expression level of rOAT-PG in the renal cortex was much higher in male rats than in female rats whereas there was no gender difference in the expression level of cyclooxygenase-2, a key enzyme producing PGE(2), and 15-PGDH in the renal cortex. Tissue PGE(2) concentration in the renal cortex was lower in male rats than in female rats, suggesting that renocortical PGE(2) concentration is primarily determined by the expression level of OAT-PG, which is regulated differently between male and female rats. Castration of male rat led to a remarkable reduction in OAT-PG expression and a significant increase in renocortical PGE(2) concentration. These alterations were recovered by testosterone supplementation. These results suggest that OAT-PG is involved in local PGE(2) clearance in the renal cortex. Although the physiological importance of the gender difference in local PGE(2) clearance is still unclear, these findings might be a key to clarifying the physiological roles of PGE(2) in the kidney.
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